The butyrophilin family genes are immunoregulatory genes with either immune stimulatory or inhibitory functions. In the present study, we analyzed three butyrophilin genes, BTN2A1 (immune-stimulatory), BTN2A2 (inhibitory), and BTNL3 (stimulatory) genes in sporadic colon cancers (CCs). By the mutation analysis, we identified the frameshift mutations of BTN2A1, BTN2A2, and BTNL3 genes in 2, 4, and 8 CCs in microsatellite instability-high (MSI-H) CCs (2.1-8.4% of MSI-H), respectively, but not the microsatellite stable (MSS) CCs. Four of 16 MSI-H CCs (25%) exhibited regional heterogeneous mutations (RHM) of BTN2A1, BTN2A2, and BTNL3 genes. In immunohistochemistry, BTNL3 expression was lost in approximately 30% of CCs, and BTN2A2 loss was minimal in CCs (around 3%) irrespective of the MSI status. Our study revered that butyrophilin family genes BTN2A1, BTN2A2, and BTNL3 harbored multiple levels of gene alterations at frameshift mutations, RHMs, and expression losses in CCs, suggesting that butyrophilin family genes could contribute to CC pathogenesis by altering immune responses.