Heat stress (HS) induces Akt/mTOR phosphorylation and FoxO3a signaling; however, whether a prior increase in heat shock protein 72 (HSP72) expression affects intracellular signaling following eccentric exercise remains unclear. We analyzed the effects of HS pretreatment on intramuscular signaling in response to acute exercise in 10‐week‐old male Wistar rats (n = 24). One leg of each rat was exposed to HS and the other served as an internal control (CT). Post‐HS, rats were either rested or subjected to downhill treadmill running. Intramuscular signaling responses in the red and white regions of the gastrocnemius muscle were analyzed before, immediately after, or 1 h after exercise (n = 8/group). HS significantly increased HSP72 levels in both deep red and superficial white regions. Although HS did not affect exercise‐induced mTOR signaling (S6K1/ERK) responses in the red region, mTOR phosphorylation in the white region was significantly higher in CT legs than in HS legs after exercise. Thr308 phosphorylation of Akt showed region‐specific alteration with a decrease in the red region and an increase in the white region immediately after downhill running. Overall, a prior increase in HSP72 expression elicits fiber type‐specific changes in exercise‐induced Akt and mTOR phosphorylation in rat gastrocnemius muscle.