The aim of the present study was to test the hypothesis that acute high-intensity interval (HIT) running induces greater activation of signaling pathways associated with mitochondrial biogenesis compared with moderate-intensity continuous (CONT) running matched for work done. In a repeated-measures design, 10 active men performed two running protocols consisting of HIT [6 × 3-min at 90% maximal oxygen consumption (V̇o2max) interspersed with 3-min recovery periods at 50% V̇o2max with a 7-min warm-up and cool-down period at 70% V̇o2max] or CONT (50-min continuous running at 70% V̇o2max). Both protocols were matched, therefore, for average intensity, duration, and distance run. Muscle biopsies (vastus lateralis) were obtained preexercise, postexercise, and 3 h postexercise. Muscle glycogen decreased ( P < 0.05) similarly in HIT and CONT (116 ± 11 vs. 111 ± 17 mmol/kg dry wt, respectively). Phosphorylation (P-) of p38MAPKThr180/Tyr182 (1.9 ± 0.1- vs. 1.5 ± 0.2-fold) and AMPKThr172 (1.5 ± 0.3- vs. 1.5 ± 0.1-fold) increased immediately postexercise ( P < 0.05) in HIT and CONT, respectively, and returned to basal levels at 3 h postexercise. P-p53Ser15 (HIT, 2.7 ± 0.8-fold; CONT, 2.1 ± 0.8-fold), PGC-1α mRNA (HIT, 4.2 ± 1.7-fold; CONT, 4.5 ± 0.9-fold) and HSP72 mRNA (HIT, 4.4 ± 2-fold; CONT, 3.5 ± 1-fold) all increased 3 h postexercise ( P < 0.05) although neither parameter increased ( P > 0.05) immediately postexercise. There was no difference between trials for any of the above signaling or gene expression responses ( P > 0.05). We provide novel data by demonstrating that acute HIT and CONT running (when matched for average intensity, duration, and work done) induces similar activation of molecular signaling pathways associated with regulation of mitochondrial biogenesis. Furthermore, this is the first report of contraction-induced p53 phosphorylation in human skeletal muscle, thus highlighting an additional pathway by which exercise may initiate mitochondrial biogenesis.