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Problem The regulatory mechanisms governing differential expression of classical major histocompatibility complex ( MHC) class I ( MHC-Ia) and non-classical MHC class I ( MHC-Ib) genes are poorly understood. Method of study Quantitative reverse transcription- polymerase chain reaction ( PCR) was used to compare the abundance of MHC-I transcripts and related transcription factors in peripheral blood mononuclear cells ( PBMC) and placental trophoblast cells ( PTC). Methylation of MHC-I CpG islands was detected by bisulfite treatment and next-generation sequencing. Demethylation of PBMC and PTC with 5′-aza-deoxycytidine was used to assess the role of methylation in gene regulation. Results MHC-I expression was higher in PBMC than PTC and was correlated with expression of IRF1, class II MHC transactivator ( CIITA), and STAT1. The MHC-Ia genes and Bo LA- NC1 were devoid of CpG methylation in PBMC and PTC. In contrast, CpG sites in the gene body of Bo LA- NC2, - NC3, and - NC4 were highly methylated in PBMC but largely unmethylated in normal PTC and moderately methylated in somatic cell nuclear transfer PTC. In PBMC, demethylation resulted in upregulation of MHC-Ib by 2.8- to 6-fold, whereas MHC-Ia transcripts were elevated less than 2-fold. Conclusion DNA methylation regulates bovine MHC-Ib expression and is likely responsible for the different relative levels of MHC-Ib to MHC-Ia transcripts in PBMC and PTC. [ABSTRACT FROM AUTHOR]