Interleukin-6 receptor-alpha (IL6R) interacts with IL6 and forms a ligand-receptor complex, which can stimulate various cellular responses, such as cell proliferation, cell differentiation, and activation of inflammatory processes. Both genetic mutation and epigenetic modification regulate gene transcription. We identified a novel splice variant of bovine IL6R, designated as IL6R-TV, which is characterized by the skipping of exon 2 of the NCBI-referenced IL6R gene (IL6R-reference). The expression levels of IL6R-TV and IL6R-reference transcripts were lower in normal mammary gland tissues. These transcripts play a potential role during inflammatory infection. We also detected two putative functional SNPs (g.19711 T > C and g.19731 G > C) located within the upstream 100 bp of exon 2. These SNPs formed two haplotypes (T-G and C-C). Two mutant pSPL3 exon-trapping plasmids (pSPL3-T-G and pSPL3-C-C) were transferred into the bovine mammary epithelial cells (MAC-T) and human embryonic kidney 293 T cells (HEK293T) to investigate the relationship between the two SNPs and the aberrant splicing of IL6R. DNA methylation levels of the alternatively spliced exon in normal and mastitis-infected mammary gland tissues were quantified through nested bisulfate sequencing PCR (BSP) and cloning sequencing. We found that DNA methylation regulated IL6R transcription. The DNA methylation level was high in mastitis-infected mammary gland tissues and stimulated IL6R expression, thereby promoting the inclusion of the alternatively spliced exon. The upregulated expression of the two transcripts was due to DNA methylation modification rather than genetic mutations.