Vaccaria segetalis has very strong effect on lactogenesis, but the mechanism is still unknown. This study aims to reveal the influence of Vaccaria segetalis on cellular lactogenesis signaling pathway of dairy cow mammary epithelial cells (DCMECs). In this study the purified DCMECs was used to screen the most effective concentration of Vaccaria segetalis water decoction for lactogenesis. DCMECs were treated with prolactin (PRL), E (estrogen) or water extraction of Vaccaria segetalis, and many lactogenesis signal molecules in DCMECs including prolactin receptor (PRLR), estrogen receptor  (ER), p-STAT5, p-mTOR, p100, GARS, PPAR and cyclinD1 were detected by using western blot assay. It was found that Vaccaria segetalis, E and PRL could promote lactogenesis of DCMECs and up-regulate these lactogenesis signal molecules except PPAR. These data reveals that Vaccaria segetalis has a similar role as E or PRL and activates cellular lactogenesis signaling pathway of DCMECs on both the translational and transcriptional levels.