This research communication investigated the role and the underlying mechanism of sn-1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6) in acetate-induced mTORC1 signaling activation and milk fat synthesis in dairy cow mammary epithelial cells. The data showed AGPAT6 knockdown significantly decreased acetate-induced phosphorylation of mTORC1 signaling molecules and intracellular triacylglycerol (TAG) content, whereas this inhibition effect was reversed after the addition of 16:0,18:1 phosphatidic acid (PA), suggesting that AGPAT6 could generate PA in response to acetate simulation, that in turn activates mTORC1 signaling. PPARγ is the upstream regulator of AGPAT6 upon acetate stimulation. Luciferase assay with clones containing various deletions and mutation in AGPAT6 promoter showed that there is a RXRα binding sequence located at −96 bp of AGPAT6 promoter. Acetate stimulation significantly increased the interaction between PPARγ and AGPAT6 via this RXRα binding site. Taken together, our data indicated that AGPAT6 could activate mTORC1 signaling by producing PA during acetate-induced milk fat synthesis, and PPARγ acts as a transcription factor to mediate the effect of acetate on AGPAT6 via RXRα.