AbstractTransforming growth factor‐β (TGF‐β) is a multifunctional growth factor, affecting cell proliferation, apoptosis, and extracellular matrix homeostasis. It also plays critical roles in mammary gland development, one of which involves inhibition of the expression of milk proteins, such as β‐casein, during pregnancy. Here we further explore the underlying signaling mechanism for it. Our results show that TGF‐β suppresses prolactin‐induced expression of β‐casein mRNA and protein in primary mouse mammary epithelial cells, but its effect on protein expression is more evident. We also find out that this inhibition is not due to the effect of TGF‐β on cell apoptosis. Furthermore, inhibition of TGF‐β type I receptor kinase activity by a pharmacological inhibitor SB431542 or overexpression of dominant negative Smad3 substantially restores β‐casein expression. By contrast, inhibition of p38 and Erk that are known to be activated by TGF‐β does not alleviate the inhibitory effect of TGF‐β. These results are consistent with our other observation that Smad but not MAPK pathway is activated by TGF‐β in mammary epithelial cells. Lastly, we show that prolactin‐induced tyrosine phosphorylation of Jak2 and Stat5 as well as serine/threonine phosphorylation of p70S6K and S6 ribosomal protein are downregulated by TGF‐β, although the former event requires considerably long exposure to TGF‐β. We speculate that these events might be involved in repressing transcription and translation of β‐casein gene, respectively. Taken together, our results demonstrate that TGF‐β abrogates prolactin‐stimulated β‐casein gene expression in mammary epithelial cells through, at least in part, a Smad3‐dependent mechanism. J. Cell. Biochem. 104: 1647–1659, 2008. © 2008 Wiley‐Liss, Inc.