MicroRNAs are one of the key determinants of muscle fibre development and phenotype in mammals. The preliminary experiment implied that microRNA‐27a (miR‐27a) might involve in regulation of muscle fibre type composition of pigs. Thereby, the present study aimed to confirm the regulatory effect of miR‐27a on porcine type I muscle fibre‐encoding gene (myosin heavy chain gene 7, MYH7) expression and its related mechanism. We firstly observed opposite expression patterns between miR‐27a and MYH7 as well as between miR‐27a and peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) during differentiation of porcine skeletal muscle satellite cells. Through the subsequent transfection analysis in porcine myotubes, we found that miR‐27a suppressed the expression of MYH7 and PGC‐1α. Besides, miR‐27a induced inhibition of PGC‐1α downstream targets, namely myocyte enhancer factor‐2C (MEF2C) along with mitochondrial biogenesis and oxidative metabolism‐related factors such as nuclear respiratory factor 1 (NRF‐1), mitochondrial transcription factor A (mtTFA), cytochrome c (Cytc) and cytochrome oxidase IV (COX Ⅳ) and succinodehydrogenase (SDH). Dual‐luciferase reporter analysis revealed that miR‐27a could bind to the predicted target site in the 3ʹ‐untranslated regions of PGC‐1α mRNA, confirming a direct targeting of PGC‐1α by miR‐27a. Moreover, PGC‐1α silencing abolished the promotive effects of miR‐27a inhibitor on MYH7, PGC‐1α and its downstream targets (MEF2C, NRF‐1, mtTFA, COX Ⅳ, Cytc and SDH) in porcine myotubes. Collectively, miR‐27a inhibits porcine MYH7 expression by negatively regulating PGC‐1α and PGC‐1α‐controlled MEF2C expression as well as mitochondrial biogenesis and oxidative metabolism. Our findings may provide a molecular target for genetic or nutritional control of muscle fibre phenotype of pigs, probably having an important implication for regulating pork quality. Summary: Skeletal muscle is a heterogeneous tissue comprising of physiologically and biochemically diverse muscle fibres, which play a key role in affecting physicochemical properties of skeletal muscle. Although it is well‐known about the transcriptional control of muscle fibre gene expression in animals through certain signalling pathways, the posttranscriptional regulation of it in pigs remains poorly understood. In this study, through bioinformatics analysis, transfection analysis and dual‐luciferase reporter analysis, we found that miR‐27a can inhibit porcine MYH7 expression by negatively regulating PGC‐1α‐controlled signalling pathways that can benefit MYH7 expression. The present study confirmed that miR‐27a can serve as a negative determinant of muscle fibre type composition aside from its reported role in affecting proliferation of skeletal muscle cells in pigs. Our findings may provide a potential target for control of type I muscle fibre formation, thus having an implication for possible regulation of muscle fibre phenotype and the associated meat quality of pigs. [ABSTRACT FROM AUTHOR]