Muscle development and lipid accumulation in muscle critically affect meat quality in livestock. Yet, the underlying genetic factors for myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we here identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle in KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and co-transfection assays showed the structural variant in the 5'-UTR of MYH3 abrogated binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of the MYH3 among worldwide pig populations indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in the porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role to improve pork quality in current breeding programs.