Successful induction of milk protein synthesis relies on prolactin/STAT5. In mice, both laminin and β1 integrin were necessary for STAT5 activity induced by prolactin treatment, resulting in transcriptional activation of β-casein. However, the mechanism by which β1 integrin increases the bovine milk protein synthesis is not well known. In order to display the crosstalk between integrin signaling and lactogenic signaling, we investigated the mechanism by which laminin mediated lactogenic effects via interaction with β1 integrin on bovine mammary epithelial cells (BMECs). Therefore, localization of β1 integrin was examined by immunofluorescence. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting. The results showed that β1 integrin were detected in basal mammary cells and basal membrane surface of adherent BMECs. However, basal distribution of β1 integrin was not sufficient to increase β-casein synthesis in the absence of integrin activation by laminin. A lactogenic hormone cocktail of insulin, hydrocortisone, and prolactin stimulated overall lactogenic effects, including upregulated expression of β1 integrin, activation of prolactin/STAT5 signaling, and consequent increase of β-casein synthesis. In response to a 24 h prolactin treatment, the abundance of STAT5, β1 integrin, and β-casein in BMECs with laminin was higher compared to that with a control substratum. Meanwhile, laminin-dependent lactogenic effects were inhibited by blocking β1 integrin function, resulting in attenuated STAT5 activity and decreased β-casein synthesis. These results indicated that β1 integrin was a key mediator of the laminin-dependent prolactin/STAT5 signaling, which regulated the sustained STAT5 activity necessary for β-casein expression in BMECs.