Bovine leukemia virus (BLV), which causes enzootic bovine leukosis, is transmitted to calves through the milk of BLV-infected dams. Bovine leukocyte antigen (BoLA)-DRB3 is a polymorphic gene associated with BLV infectivity and proviral load (PVL). However, the effect of BoLA-DRB3 polymorphism on the infectivity and PVL of milk from BLV-infected dams remains unknown. This study examined milk from 259 BLV-infected dams, including susceptible dams carrying at least one BoLA-DRB3*012:01 or *015:01 allele with high PVL, resistant dams carrying at least one BoLA-DRB3*002:01, *009:02, or *014:01:01 allele with low PVL, and neutral dams carrying other alleles. The detection rate of BLV provirus and PVL were significantly higher in milk from susceptible dams than in that from resistant dams. This result was confirmed in a three-year follow-up study in which milk from susceptible dams showed a higher BLV provirus detection rate over a longer period than that from resistant dams. The visualization of infectivity of milk cells using a luminescence syncytium induction assay showed that the infectious risk of milk from BLV-infected dams was markedly high for susceptible dams compared to resistant ones. This is the first report confirming that BoLA-DRB3 polymorphism affects the PVL and infectivity of milk from BLV-infected dams.