Milk fat synthesis in dairy cows can be inhibited by unique fatty acid intermediates that are produced during rumen biohydrogenation. One of these inhibitory intermediates is trans-10, cis-12 conjugated linoleic acid (CLA), and this milk fat depression (MFD) involves a coordinated decrease in mammary expression of lipogenic enzymes. We investigated the sterol response element binding protein (SREBP) transcription factor system in the mammary tissue of cows during MFD, which was induced by a low forage, high oil (LF/HO) diet and trans-10, cis-12 CLA infusion. The LF/HO diet and CLA treatment decreased milk fat yield by 38 and 24%, respectively. Treatments causing MFD decreased expression of SREBP1 and the insulin responsive gene (INSIG) 1, consistent with decreased abundance of active SREBP1. The LF/HO diet also decreased expression of INSIG2 and SREBP cleavage activating protein. In addition, we identified the involvement of thyroid hormone responsive spot 14 (S14) in the regulation of mammary synthesis of milk fat. A broader role for S14 in the trans-10, cis-12 CLA-mediated decrease in fat synthesis was explored by mining publicly available microarray datasets, and we found that mouse adipose expression of S14 was decreased in response to CLA treatment. Overall, the decreased mammary expression of SREBP1, SREBP activation protein, and the coordinated reduction in SREBP1-responsive lipogenic enzymes provides strong support for a central role of SREBP1 in the regulation of milk fat synthesis. In addition, our results provide evidence for an involvement of S14 in mammary regulation of milk fat synthesis and a possible broader role for S14 in the reported antiobesity effects of CLA.