We investigated if acute endurance-type exercise interacts with insulin-stimulated activation of atypical protein kinase C (aPKC) and insulin signalling to peptide chain elongation in human skeletal muscle. Four hours after acute one-legged exercise, insulin-induced glucose uptake was ∼80% higher (N= 12, P < 0.05) in previously exercised muscle, measured during a euglycaemic–hyperinsulinaemic clamp (100 μU ml−1). Insulin increased (P < 0.05) both insulin receptor substrate (IRS)-1 and IRS-2 associated phosphatidylinositol (PI)-3 kinase activity and led to increased (P < 0.001) phosphorylation of Akt on Ser473 and Thr308 in skeletal muscle. Interestingly, in response to prior exercise IRS-2-associated PI-3 kinase activity was higher (P < 0.05) both at basal and during insulin stimulation. This coincided with correspondingly altered phosphorylation of the extracellular-regulated protein kinase 1/2 (ERK 1/2), p70S6 kinase (P70S6K), eukaryotic elongation factor 2 (eEF2) kinase and eEF2. aPKC was similarly activated by insulin in rested and exercised muscle, without detectable changes in aPKC Thr410 phosphorylation. However, when adding phosphatidylinositol-3,4,5-triphosphate (PIP3), the signalling product of PI-3 kinase, to basal muscle homogenates, aPKC was more potently activated (P= 0.01) in previously exercised muscle. Collectively, this study shows that endurance-type exercise interacts with insulin signalling to peptide chain elongation. Although protein turnover was not evaluated, this suggests that capacity for protein synthesis after acute endurance-type exercise may be improved. Furthermore, endurance exercise increased the responsiveness of aPKC to PIP3 providing a possible link to improved insulin-stimulated glucose uptake after exercise.