This study investigated the effect of regular postexercise cold water immersion (CWI) on muscle aerobic adaptations to endurance training. Eight males performed 3 sessions/wk of endurance training for 4 wk. Following each session, subjects immersed one leg in a cold water bath (10°C; COLD) for 15 min, while the contralateral leg served as a control (CON). Muscle biopsies were obtained from vastus lateralis of both CON and COLD legs prior to training and 48 h following the last training session. Samples were analyzed for signaling kinases: p38 MAPK and AMPK, peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), enzyme activities indicative of mitochondrial biogenesis, and protein subunits representative of respiratory chain complexes I–V. Following training, subjects' peak oxygen uptake and running velocity were improved by 5.9% and 6.2%, respectively ( P < 0.05). Repeated CWI resulted in higher total AMPK, phosphorylated AMPK, phosphorylated acetyl-CoA carboxylase, β-3-hydroxyacyl-CoA-dehydrogenase and the protein subunits representative of complex I and III ( P < 0.05). Moreover, large effect sizes (Cohen's d > 0.8) were noted with changes in protein content of p38 ( d = 1.02, P = 0.064), PGC-1α ( d = 0.99, P = 0.079), and peroxisome proliferator-activated receptor α ( d = 0.93, P = 0.10) in COLD compared with CON. No differences between conditions were observed in the representative protein subunits of respiratory complexes II, IV, and V and in the activities of several mitochondrial enzymes ( P > 0.05). These findings indicate that regular CWI enhances p38, AMPK, and possibly mitochondrial biogenesis.