The intramuscular fat (IMF), fatty acid and amino acid compositions of pork are intricately linked to meat quality, flavor profile, and nutritional composition, and have potential implications for human health. Lipid accumulation in pork is initiated by the biosynthesis of fatty acids and regulated by a complex network of genes. In this study, the IMF content and genotyping of large-scale slaughtered Yorkshire pigs were assessed. Transcriptome sequencing of muscles from 17 individuals and fatty and amino acid analyses of muscles from 28 individuals according to IMF content were conducted. Phenotypic analysis showed a high correlation between IMF and most fatty acids, and the composition ratio of different types of fatty acids varied with IMF content. A negative correlation between the n-6/n-3 polyunsaturated fatty acid (PUFA) ratio and increase in IMF content significantly enhanced the levels of essential fatty acids and ameliorated the n-6/n-3 PUFA ratio in pork, thereby elevating its nutritional value to better align with contemporary health standards. A comprehensive analysis that integrated a genome-wide association study, differential gene expression analysis, and weighted gene co-expression network analysis was employed to identify the regulatory mechanisms of lipids. PRLR, SEC11C, ALPK2, CPLX4, APC, and CREB5 were identified as key candidate genes that affect intramuscular lipids and fatty acids. Through molecular and cellular experiments, our results indicated that high APC and CREB5 gene expression significantly promotes lipogenesis in cells, where these genes play an important role in regulatory pathways related to lipid synthesis in animals, which may affect fat deposition and fatty acid composition in pork. Overall, these results lay the foundation for an in-depth analysis of the genetic regulation of pork lipids and nutrition, and also provide molecular regulatory markers for the primary selection of pigs with better meat quality.