Galectin-9 levels have been reported to be altered in several cancer types, but the mechanism that regulates its expression has not been clarified. Galectin-9 is encoded by the LGALS9 gene, which gives rise to eight mRNA variants. The aims of the study were (i) to identify the mRNA variants of LGALS9, (ii) to characterize CpG methylation and H3K9 and H3K14 histone acetylation at the promoter of the LGALS9 gene and (iii) to characterize the relationship between these modifications and LGALS9 expression level in cervical cancer cells. All mRNA variants were detected in HaCaT (nontumoural keratinocytes) and SiHa cells, and seven were observed in HeLa cells. The promoter region of LGALS9 contains eight CpG dinucleotides. No hypermethylation pattern related to low LGALS9 expression was identified in tumour cells. ChIP analysis demonstrated higher acetylation of H3K9ac and H3K14ac in HaCaT cells, which was related to higher mRNA levels. The presence of the mRNA variants suggests that alternative splicing may regulate the expression of galectin-9 isoforms. The results of this study suggest that histone acetylation but not promoter CpG methylation may be involved in the transcriptional regulation of the LGALS9 gene.