The effects of ABO incompatibility on hematopoietic stem cell transplantation remain controversial. Large cohorts are required to obtain findings that allow for definite conclusions. We previously demonstrated poor overall survival and increased treatment-related mortality (TRM) in ABO-incompatible unrelated bone marrow transplantation (UR-BMT) performed during the period from 1993 to 2005. To improve our understanding of ABO-incompatible transplantation, we reanalyzed the effects of ABO mismatch in a UR-BMT cohort in Japan after 2000. Multivariate analyses for the 2000-2006 cohort showed that major ABO mismatch was associated with poor overall survival (HR, 1.211; 95% CI, 1.062 to 1.381; p = 0.004) and increased TRM (HR, 1.357; 95% CI, 1.146 to 1.608; p < 0.001). In the 2007-2015 cohort, major incompatibility had no effect on overall survival (HR, 0.987, p = 0.804) or TRM (HR, 1.020, p = 0.790). Delayed engraftment of erythrocytes, platelets, and neutrophils in cases of major mismatch was common between the two cohorts. In conclusion, the adverse effect of ABO major incompatibility has become less significant over time.