Abstract. Fatty acid composition is one of the main factors affecting health benefits of food. Stearoyl-CoA desaturase 1 (SCD), acetyl-CoA carboxylase alpha (ACACA) and lipoprotein lipase (LPL) have been considered as the rate-limiting enzymes in the biosynthesis of different fatty acids critical in lipid metabolism. The aim of our study was the analysis of differences in expression profiles of three ovine genes related to lipid metabolism (LPL, ACACA, SCD) depending on feeding system and tissue type. The gene expression measurement was performed using a real-time PCR method on 60 old-type Polish Merino Sheep, which were divided into three feeding groups (I – complete pellet mixture, n =  12; II – complete mixture with addition of fresh grass, n =  24; III – complete mixture with addition of fresh red clover, n =  24). From all lambs, tissue samples – subcutaneous fat, perirenal fat and liver – were collected immediately after slaughter and LPL, ACACA and SCD expression was estimated based on two endogenous controls (RPS2 – ribosomal protein S2; ATP5G2 – H(+)-transporting ATP synthase). Our research indicated that supplementation of diet with an addition of fresh grass or red clover significantly (P < 0.05) decreased the expression of SCD, ACACA and LPL genes in fat tissue compared to standard complete pelleted mixture. On the other hand, the highest expression of ACACA was detected in liver tissue collected from sheep fed a diet with an addition of fresh red clover (P < 0.05). In turn, the highest expression of the SCD gene was detected in animals fed with grass supplementation (P < 0.05). Regardless of diet supplementation, the highest SCD transcript abundance was detected in perirenal fat, while LPL and ACACA expression was the highest in both perirenal and subcutaneous fat. The ability of nutrigenomic regulation of transcription of analyzed genes confirmed that these genes play a critical role in regulation of lipid metabolism processes in sheep and could be associated with fatty acid profiles in milk and meat.