Lipid supplementation with palmitic acid (C16:0) has been shown to increases the milk yield and milk fat content. Also, changes in the fatty acid profile have been shown variable reducing or not short and medium-chain fatty acid from the de novo synthesis pathway. However, the molecular mechanisms have not been fully investigated. The objective of this study was to evaluate the effect of palmitic acid (C16:0) on the expression of lipogenic genes involved in the milk fat synthesis [(acetyl CoA carboxylase α; ACC-α) isoforms from promoters II and III and fatty acid synthase (FASN)], fatty acid internalization (CD36 molecule; CD36), intracellular transport of fatty acids (fatty acid binding protein; FABP 3 and 4) and fatty acid desaturation (stearoil CoA desaturase; SCD). Mammary explants weighing 30 ± 0.005 mg from 3 Lacaune ewes with 40 ± 6 DIM and BCS of 3.25, obtained through biopsies were cultured in sextuplicates for 24h with one of the following treatments: Control [culture medium + bovine albumin (98%; BSA fatty acid free) at 75 μM] and Palmitic Acid [culture medium + C16:0 (99%) at 75 μM]. Subsequently, total RNA was extracted, complementary DNA (cDNA) was synthesized and the real-time quantitative PCR analysis (RT-qPCR) carried out. The data were analyzed using the MIXED procedure of SAS assuming treatment as a fixed effect and the explant as random. Palmitic acid increased (P < 0.05), respectively, ACC-α PIII, ACC-α PII, and FASN gene expression 1.5-, 1.3-, and 1.3-fold compared with the Control. However, palmitic acid did not stimulate the gene expression of CD36, FABP3, FABP4, and SCD. According the literature, the observed increase in the gene expression of the de novo pathway lipogenic genes suggests that short and medium-chain fatty acids are needed to be esterified on the triacylglycerol for preserving milk fat fluidity.