Trans-10, cis-12 conjugated linoleic acid (CLA) is a fatty acid intermediate produced during rumen biohydrogenation of dietary polyunsaturated fatty acids in the cow. Trans-10, cis-12 CLA inhibits de novo milk fat synthesis in the mammary gland through a coordinated downregulation of key lipogenic enzymes and the nuclear protein thyroid hormone responsive spot 14 (S14). A previous study using a mouse model showed that the S14 knockout (KO) mouse was hyperresponsive to CLA. In KO mice, mammary lipogenesis decreased by 82% in response to a 5-day treatment of 20 mg/d trans-10, cis-12 CLA, whereas in wildtype (WT) controls, rates of lipogenesis decreased by 23%. The objective of the present study was to investigate whether the KO mouse was also hypersensitive to CLA. This was done by testing the lactogenic response of the KO mice to a low dose of CLA. WT and KO C57BL/6J mice were orally administered with either water (control) or 3.5 mg/d of trans-10, cis-12 CLA for 5 days. In response to CLA treatment, KO dams had a 28.7% reduction in the percentage of de novo synthesized fatty acids in milk, whereas in WT dams this reduction (8.9%) was not significant, and there was an interaction of genotype and CLA treatment. Investigations with mammary explants demonstrated that lipogenic capacity decreased in both KO and WT dams, but there was no interaction of genotype and CLA treatment. A compilation of results from the present study and from previous studies suggests that the ED50 of CLA of the WT and KO mice are similar, thus indicating that the sensitivity of the two genotypes are similar. Therefore, the S14 KO mouse is hyperresponsive to CLA-induced inhibition of mammary lipogenesis but sensitivity is unaltered.