Objective: To explore the changing pattern of lactose content during mammary gland development of Holstein cows. Methods: High-performance liquid chromatography was used to determine lactose content. Results: Lactose was detectable in the mammary gland during the perinatal period, and showed an increasing trend during the lactation period, reaching a peak after 140 d of lactation, and then a dramatic decrease during the involution period, reaching a very low level after 30 d of involution. In conclusion, lactose content exhibits different levels in the mammary gland of Holstein cows during different physiological periods.