Signal transducer and activator 5 (STAT5) plays important roles in regulating mammary glandular cell proliferation and milk-protein gene expression. However， the functions of STAT5a and STAT5b genes in lactation of buffalo remain uninvestigated. In this study， full-length STAT5a (2502 bp) and STAT5b (2515 bp) coding sequences were isolated for the first time. The highest STAT5a gene expression was found in buffalo mammary glands and the highest STAT5b gene expression was found in buffalo brains and mammary glands. H&E staining indicated that STAT5a and STAT5b are mainly expressed in epithelial cells of buffalo mammary glands. Next， we investigated the functions of STAT5 by knocking down and overexpressing STAT5 in buffalo mammary epithelial cells (BuMECs). According to our results， STAT5 knockdown resulted in inhibited G1/S transition of BuMECs and significantly lower expression of milk-protein genes， whereas overexpression of STAT5 resulted in significantly higher expression of milk-protein genes. In summary， our results demonstrate that STAT5 can regulate the cell cycle transition of BuMECs and affect the expression of milk-protein genes. Our research lays a foundation for further study of the role of STAT5 in mammary gland development and lactation.