The objective of this study was to characterize the mRNA expression in subcutaneous adipose tissue (AT) via RTqPCR of genes related to fatty acid (FA) synthesis and desaturation (ACACA, FADS2, FASN, SCD1); regulation of transcription (INSIG1, SCAP, SREBF1, THRSP, PPARGC1A); lipid droplet formation (PLIN2, SLC27A6); triacylglycerol synthesis (DGAT1, DGAT2, LPIN1); FA import into cells (LPL, VLDLR); and intracellular transport (ACSL1, ACSL2, FABP3, FABP4) in dairy cows supplemented with unsaturated (olive oil; OO) and saturated (hydrogenated vegetable oil; HVO) lipids. Fifteen cows averaging 189 ± 28 d in milk (average ± SD) at the beginning of the study were randomly assigned to treatment groups. During 9 wk animals were fed a control diet with no added lipid (n = 5 cows; basal diet), and fat-supplemented diets containing OO (n = 5 cows; 30 g/kg DM) and HVO (n = 5 cows; 30 g/kg DM). AT was obtained from the tail-head area at the onset of the study (P0) and after 9 wk of supplementation. Compared with control and HVO, OO increased (P < 0.05) milk yield, and reduced (P < 0.05) milk fat yield and milk somatic cell counts. Relative expression was determined using P0 as a reference condition. Relative mRNA expression was determined using the Pair Wise Fixed Reallocation Randomization Test built in REST (2008) using 5000 iterations. Correction and normalization to the reference genes (GAPDH, UXH, EIF3K) were calculated using the Ct values for each sample as the input variable. OO upregulated (P < 0.05) the expression of ACACA, PLIN2, THRSP, DGAT1, LPL, and FABP4. HVO upregulated (P < 0.05) the expression of SLC27A6. Overall, OO upregulated some genes related to FA metabolism in adipose tissue whereas HVO induced upregulation on a gene related to FA import. Our results suggest that unsaturated lipid sources may have stronger lipogenic effects in bovine AT than saturated sources in long-term supplementation. This study provides further knowledge on FA metabolism in adipose tissue and data can be used to develop new strategies for a better nutritional management in dairy cows. This study was sponsored by a research grant from FONDECYT 1170400, Chile.