Beneficial microRNAs (miRNAs) are highly expressed microRNAs (miRNAs) in milk, such as mir148a-3p, which was linked to immune system development and disease prevention. To better understand their expression and secretion regulation mechanisms in breast milk, more research was needed. Researchers believed that oxytocin was involved in regulating milk-derived miRNA expression and secretion. The influence of oxytocin on miRNA expression in human mammary cells was first investigated. Second, before, during, and after labor, the expression of specific miRNAs in mothers’ colostrum treated or not with oxytocin was compared. Third, quantitative real-time PCR was used to examine MiRNA expression. When oxytocin-treated mammary cells and their released extracellular vesicles to the medium were compared to untreated cells, miR-148a expression was dramatically increased while miR-320 expression was significantly downregulated. In the human colostrum of exogenous oxytocin-treated mothers, miR-148a was elevated, while miR-320 was shown to be downregulated. Furthermore, when compared to miR-148a, miR-320 was strongly expressed in the colostrum of moms who did not receive exogenous oxytocin. In comparison to miR-320, the expression of miRNA-148-3p was strongly expressed in the milk of women who received exogenous oxytocin. Oxytocin regulates the expression of primary milk-derived miRNAs, according to the study. The outcomes modulated miRNA expression shed new light on oxytocin’s role in milk composition. The results suggested that oxytocin promotes miRNA expression in mammary epithelial cells and human milk, changing the composition of human milk and perhaps contributing to newborn health.