Due to its rich nutritional value, yak milk is an important food source in the alpine pastoral areas. However, yaks have a low milk yield. The Hippo pathway participates in cell proliferation and organ development. We aimed to determine the regulatory mechanism of this pathway in yak mammary cells. A greater understanding of how the expression of its essential genes influence the reproductive cycle could lead to improvements in lactation performance. The expression levels of the key genes MST1, LATS1, YAP1, and TEAD1 were detected by quantitative real-time PCR, Western blotting, and immunohistochemistry during the growth, lactation, and dry periods (GP, LP and DP, respectively). The MST1 and LATS1 mRNA and protein expression level was highest during GP and lowest during LP. The YAP1 and TEAD1 mRNA and protein expression level decreased from GP to LP and DP. MST1 and LATS1 were expressed in the cytoplasm whereas YAP1 and TEAD1 were expressed in the nucleus and cytoplasm, respectively. The differential expression of MST1, LATS1, YAP1, and TEAD1 expression in the yak mammary gland during different developmental stages strongly suggests that they play an important role in the regulation of developmental functions under different physiological conditions.