Myostatin (MSTN) is a member of the transforming growth factor-β superfamily that negatively regulates skeletal muscle development. A lack of MSTN induces muscle hypertrophy and increases formation of fast-twitch (Type II) muscle fibres. This study investigated muscle development in newborn heterozygous (MSTN+/-) and homozygous (MSTN-/-) MSTN-knockout piglets. Detailed morphological and gene and protein expression analyses were performed of the biceps femoris, semitendinosus and diaphragm of MSTN+/-, MSTN-/- and wild-type (WT) piglets. Haematoxylin-eosin staining revealed that the cross-sectional area of muscle fibres was significantly larger in MSTN-knockout than WT piglets. ATPase staining demonstrated that the percentage of Type IIb and IIa muscle fibres was significantly higher in MSTN-/- and MSTN+/- piglets respectively than in WT piglets. Western blotting showed that protein expression of myosin heavy chain-I was reduced in muscles of MSTN-knockout piglets. Quantitative reverse transcription-polymerase chain reaction revealed that, compared with WT piglets, myogenic differentiation factor (MyoD) mRNA expression in muscles was 1.3- to 2-fold higher in MSTN+/- piglets and 1.8- to 3.5-fold higher MSTN-/- piglets (P<0.05 and P<0.01 respectively). However, expression of myocyte enhancer factor 2C (MEF2C) mRNA in muscles was significantly lower in MSTN+/- than WT piglets (P<0.05). MSTN plays an important role in skeletal muscle development and regulates muscle fibre type by modulating the gene expression of MyoD and MEF2C in newborn piglets.