Differences in muscle fiber development between Tibetan and Yorkshire pigs determine their performance in terms of meat quality, taste, and food value. To investigate the molecular regulation of muscle fiber development by fibronectin type III domain-containing protein 5 (FNDC5), a preliminary study of FNDC5 gene expression in the longissimus dorsi muscle and Leg muscle tissues of Tibetan and Yorkshire Pigs was conducted using RT–qPCR and western blotting. Muscle tissues were sectioned and stained to observe the muscle fiber diameter and area under a microscope. The mRNA and protein expression levels of FNDC5, and the diameter and area of muscle fibers in the longissimus dorsi muscle and Leg muscle tissues of Tibetan pigs were significantly lower than those in Yorkshire Pigs. The results imply that FNDC5 negatively regulates muscle fiber diameter in pigs. The role of FNDC5 in pork quality has important implications for improving pork production efficiency, pork quality, and the sustainable development of the livestock industry.