Skeletal muscle has numerous nuclei within a cell. The nucleus is considered as the central organelle for muscle protein synthesis (MPS). However, it is unclear whether myonuclear number is associated with MPS capacity within the individual muscle fibres. Therefore, the purpose of the present study was to reveal the relationship between myonuclear number per unit muscle fibre length and MPS under basal and conditions of elevated MPS by high-intensity muscle contraction (HiMC) using an in vivo nascent protein labelling technique (SUnSET) in rodents. We found that myonuclear number was positively correlated with MPS in individual muscle fibres in the basal condition. Similarly, ribosomal protein S6 (rpS6) content, which is a rough estimate of ribosome content, was positively correlated with MPS. However, myonuclear number was not associated with rpS6 content. In contrast to the basal condition, when MPS was increased by acute HiMC, no correlation was observed between myonuclear number and MPS, but the association between rpS6 and MPS was maintained. Importantly, these observations indicate that the number of nuclei in individual myofibers is related only to MPS at rest. However, the ribosome content in individual fibres is related to MPS of individual myofibers both at rest and following HiMC.