The presence of post‐translational regulation of MHC class II (MHC II) under physiological conditions has been demonstrated recently in dendritic cells (DCs) that potently function as antigen‐presenting cells (APCs). Here, we report that MARCH‐I, an E3 ubiquitin ligase, plays a pivotal role in the post‐translational regulation of MHC II in B cells. MARCH‐I expression was particularly high in B cells, and the forced expression of MARCH‐I induced the ubiquitination of MHC II. In B cells from MARCH‐I‐deficient mice (MARCH‐I KO), the half‐life of surface MHC II was prolonged and the ubiquitinated form of MHC II completely disappeared. In addition, MARCH‐I‐deficient B cells highly expressed exogenous antigen‐loaded MHC II on their surface and showed high ability to present exogenous antigens. These results suggest that the function of MHC II in B cells is regulated through ubiquitination by MARCH‐I.