The level of n-6 and n-3 polyunsaturated fatty acids may affect many cellular systems and functions via nuclear receptors or the bioactive lipid regulation of gene expression. The objective of this study was to investigate the changes in the muscle transcriptome and the biological functions regulated by increased consumption of n-3 and n-6 fatty acids in the pig gluteus medius muscle. The transcriptome of the gluteus medius muscle was studied for pigs subjected to either a control diet or a diet supplemented with linseed and rapeseed oil to increase polyunsaturated fatty acid content. Next-generation sequencing (NGS) was used to generate the muscle tissue transcriptome database. The results of the NGS indicate a role of the fatty acids in the regulation of the expression of genes essential for muscle tissue development and functioning. Functional analysis revealed that the identified genes were important for a number of biological processes including protein development, signaling, inflammatory response, membranes, and lipid metabolism. The genes that were expressed specifically in only one of the dietary groups were responsible for many of the same processes listed above, indicating that biological processes were strongly modulated and tended to switch between coherent stages in response to increased levels of n-6 and n-3 fatty acids. Several nuclear receptors regulating transcription showed altered expression, including hepatocyte nuclear factor-4α (HNF4alpha), carbohydrate responsive element binding protein (ChREBP), and nuclear factor κB (NFkappaΒ). We suggest that the identified changes were largely due to regulation of those transcription factors. Our results provide strong evidence that n-6 and n-3 fatty acids regulate fundamental metabolic processes in muscle tissue development and functioning.