Abstract Male muskrats have one pair of scented glands that grow and involute annually. To investigate the annual changes in the scented gland, we measured the expressions of aromatase cytochrome P450 (P450arom) and estrogen receptors (ERs) in the scented glands. P450arom was expressed in glandular cells and epithelial cells in the scented glands during the breeding season, and only in glandular cells during the non-breeding season. ERα and ERβ were also detected in different types of cells in the scented gland during the breeding and non-breeding seasons. Both mRNA and protein levels of P450arom, ERα and ERβ were higher in the scented glandular tissues during the breeding season than those during the non-breeding season. In addition, small RNAs sequencing showed that the predicted targets of the significantly changed microRNAs (miRNA) might be the genes encoding P450arom and ERs. In conclusion, the seasonal changes in the expression of P450arom and estrogen receptors may be involved in the regulation of scented gland functions.