The objective of this study was to test the hypothesis that stearic acid (SA) supplementation increases milk fat content and overcomes the antilipogenic effects of trans-10, cis-12 conjugated linoleic acid (CLA) in lactating ewes. Twenty-eight Lacaune ewes (36 (sd 2) days in lactation; 70·5 (sd) 9·6 kg of body weight), producing 1·8 (sd 0·4) kg of milk/d, were used in a completely randomised design (seven ewes/treatment) for 21 d. The treatments were: (1) Control; (2) CLA (6·4 g/d of trans-10, cis-12 CLA); (3) SA (28 g/d of SA) and (4) SA in association with trans-10, cis-12 CLA (CLASA; 6·4 g/d of trans-10, cis-12 CLA plus 28 g/d of SA). All data were analysed using a mixed model that included the fixed effect of treatment and the random effect of ewe. SA did not alter milk fat content and yield relative to Control (91·9 v. 91·2 (sd 4·1) g/d). CLASA was not able to overcome the reduction in fat content and fat yield induced by CLA (75 v. 82 (sd 0·14) g/d). SA increased the relative abundance of CD36, fatty acid-binding protein 4 (FABP4) and PPAR-γ mRNA by 140, 112 and 68 % compared with CLASA. SA also reduced the relative abundance of acetyl-CoA carboxylase α promoter II and stearoyl-CoA desaturase (SCD) when compared with Control (45 and 39 %). Compared with CLA, CLASA treatment had no effect on the mRNA abundance of fatty acid synthase, lipoprotein lipase, CD36, SCD, FABP4, acylglycerolphosphate acyltransferase 6, sterol regulatory element-binding protein 1 and PPAR-γ. In conclusion, SA supplementation did not increase milk fat synthesis and did not overcome the CLA-induced milk fat depression when associated with trans-10, cis-12 CLA.