For fans of One Piece, very few characters are as beloved as Roronoa Zoro. Introduced at the end of the very first episode of the anime, Pirate Hunter Zoro has been a staple of the series for practically as long as Luffy has. Conversely, his sense of humor wasn't nearly as long-lasting. Regardless of how strong he has become or how cool his fights have been, many fans miss the days when they saw him crack jokes, not just heads.
The fact of the matter is that over time, Zoro went from a fun-loving pirate to a much more stoic warrior. Of course, there isn't just one reason for this change, but multiple ones that have honed the character into the badass he is today, much as Zoro has had to hone his swordsmanship skills.
Since the beginning of the series and even in his own flashbacks, Zoro was portrayed as practically unbeatable, with his late friend Kuina being the only person he ever lost to. After her death, he dedicated himself even further to the sword, choosing to honor her memory by fulfilling their shared dream of becoming the greatest swordsman alive. To his credit, he largely succeeded. Throughout the East Blue, he became notorious for being an unstoppable "demon" who all pirates feared. However, this changed somewhat during the Baritie arc.
Over the course of the East Blue saga's Baratie arc, Zoro underwent his first big change due to his iconic battle with Dracule Mihawk. Zoro's dream since the start of the series has been to become the "World's Greatest Swordsman," with this desire being the core aspect of his character arc as well. Unfortunately, the only way to prove this is to beat the current titleholder in a sword fight. As fans will know, Zoro lost this duel and almost his own life, although not before he was able to at least impress Mihawk with his tenacity.
After the duel marks one of Zoro's most important character moments, where he proclaims that the King of Pirates needs a strong, deserving crew and vows to Luffy that he'll never lose again. It's important to note this moment, as not only is it the first time fans saw Zoro lose a fight, but it's also very much implied that it was the only time he had ever lost since setting out on his journey. Before the duel, Zoro could be seen on many occasions cracking jokes and laughing with Luffy and the rest of the original Straw Hats, but after his overwhelming defeat, this all seemed to change.
The loss to Mihawk humbled Zoro in a way he hadn't been since Kuina was alive. Regardless of how much stronger he was compared to the vast majority of the people he knew, deep down, he was aware of just far the gap was between him and his ultimate goal. This humbling affected Zoro on a deep level, so much so that it seems to have caused him to become much more serious. His arrogance and unearned confidence are what led him to believe that he stood a chance against Mihawk, and in many ways, every fight since has further proved to him just how far apart they are. If he could barely beat Daz Bones and Kaku, there's no chance he could beat Mihawk.
The gap between the two was further established throughout the Thriller Bark and Sabaody Archipelago arcs. In the former, he is beaten handily by Bartholomew Kuma, barely surviving their encounter, while in the latter, he is beaten by Admiral Kizaru with seemingly no effort. If it weren't for Kuma returning to secretly save him and the other Straw Hats, they would have all surely died, with Zoro having no way of stopping him.
This feeling of helplessness is further exacerbated after finding out about Portgas D. Ace's death during the Marineford arc. Seeing that his captain's brother died, someone who even Zoro acknowledged as incredibly strong, as well as realizing just how much pain it caused Luffy, made Zoro realize just how high the stakes had become. Realizing he could no longer stay as weak as he was, he dedicated himself to his training during the two-year timeskip, going as far as to swallow his pride and beg Mihawk to train him to become stronger.
So, for fans who wonder why Zoro stopped laughing, it isn't as simple as merely losing his sense of humor along with his sense of direction. Rather, it was due to Zoro realizing just what his crew needed him to be, as well as what he needed to become, in order to accomplish his own goals. In his eye(s), the King of Pirates needs and deserves the World's Greatest Swordsman -- a responsibility that Zoro knows he needs to take seriously.
