Mucosal-associated invariant T (MAIT) cells are a type of innate lymphocyte and recognize riboflavin (vitamin B2) synthesis products presented by MHC-related protein 1. We investigated long-term reconstitution of MAIT cells and its association with chronic graft-versus-host disease (cGVHD) in a cross-sectional cohort of 173 adult patients after allogeneic hematopoietic cell transplantation. According to donor source, the number of MAIT cells significantly correlated with time after cord blood transplantation (CBT) but not with time after bone marrow transplantation or peripheral blood stem cell transplantation. The number of MAIT cells was significantly lower in patients with cGVHD compared with patients without cGVHD. We also examined the association between MAIT cell reconstitution and gut microbiota as evaluated by 16S ribosomal sequencing of stool samples 1 mo post-CBT in 27 adult patients undergoing CBT. The diversity of gut microbiota was positively correlated with better MAIT cell reconstitution after CBT. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States analysis indicated that amounts of ribB and ribA genes were significantly higher in the microbiomes of patients with subsequent MAIT cell reconstitution after CBT. In conclusion, long-term MAIT cell reconstitution is dependent on the type of donor source. Our data also unveiled an important role for the interaction of circulating MAIT cells with gut microbiota in humans.