Apoptosis has been implicated in the regulation of denervation‐induced muscle atrophy. However, the activation of apoptotic signal transduction during muscle denervation has not been fully elucidated. The present study examined the apoptotic responses to denervation in rat gastrocnemius muscle. Following 14 days of denervation, the extent of apoptotic DNA fragmentation as determined by a cytosolic nucleosome ELISA was increased by 100% in the gastrocnemius muscle. RT‐PCR and immunoblot analyses indicated that Bax was dramatically upregulated while Bcl‐2 was modestly increased; however, the Bax/Bcl‐2 ratio was significantly increased in denervated muscles relative to control muscles. Analyses of ELISA and immunoblots from mitochondria‐free cytosol extracts showed a significant increase in mitochondria‐associated apoptotic factors, including cytochrome c, Smac/DIABLO and apoptosis‐inducing factor (AIF). In addition to the upregulation of caspase‐3 and ‐9 mRNA, pro‐/cleaved caspase protein and proteolytic activity levels, the X‐linked inhibitor of apoptosis (XIAP) protein level was downregulated. The cleaved product of poly(ADP‐ribose) polymerase (PARP) was detected in muscle samples following denervation. Although we did not find a difference in the inhibitor of DNA binding/differentiation‐2 (Id2) and c‐Myc protein contents between the denervated and control muscles, the protein content of tumour suppressor p53 was significantly increased in both the nuclear and the cytosolic fractions with denervation. Moreover, denervation increased the protein content of HSP70, whereas the MnSOD (a mitochondrial isoform of superoxide dismutase) protein content was diminished, which indicated that denervation might have induced cellular and/or oxidative stress. Our data show that mitochondria‐associated apoptotic signalling is upregulated during muscle denervation. We interpret these findings to indicate that apoptosis has a physiologically important role in regulating denervation‐induced muscle atrophy.