In our previous genome-wide association study on milk production traits in Chinese Holstein cows, HSF1 (heat shock factor 1) and CPSF1 (cleavage and polyadenylation specific factor 1) were found to be strongly associated with milk fat and protein percentages. However, their roles in milk fat and protein synthesis and the underlying mechanism are still largely unknown. In this study, we verified the effects of their expressions on milk fat and milk protein synthesis in MAC-T cells. We showed that HSF1 can participate in the AKT/mTOR signaling pathway, one of the most important pathways for fat and protein synthesis, through its interaction with the AKT protein and influence the downstream genes in this pathway to regulate milk fat and milk protein synthesis. We also found that HSF1, as a transcription factor, can bind to the promoter region of CPSF1 to regulate its transcription and expression, which in turn modulates the expression of SREBP1 and thereby influences the synthesis of milk fat.