Phospholemman (PLM, FXYD1) is a partner protein and regulator of the Na(+)-K(+)-ATPase (Na(+)-K(+) pump). We explored the impact of acute and short-term training exercise on PLM physiology in human skeletal muscle. A group of moderately trained males (n = 8) performed a 1-h acute bout of exercise by utilizing a one-legged cycling protocol. Muscle biopsies were taken from vastus lateralis at 0 and 63 min (non-exercised leg) and 30 and 60 min (exercised leg). In a group of sedentary males (n = 9), we determined the effect of a 10-day intense aerobic cycle training on Na(+)-K(+)-ATPase subunit expression, PLM phosphorylation, and total PLM expression as well as PLM phosphorylation in response to acute exercise (1 h at ∼72% Vo(2peak)). Biopsies were taken at rest, immediately following, and 3 h after an acute exercise bout before and at the conclusion of the 10-day training study. PLM phosphorylation was increased both at Ser(63) and Ser(68) immediately after acute exercise (75%, P < 0.05, and 30%, P < 0.05, respectively). Short-term training had no adaptive effect on PLM phosphorylation at Ser(63) and Ser(68), nor was the total amount of PLM altered posttraining. The protein expressions of α(1)-, α(2)-,and β(1)-subunits of Na(+)-K(+)-ATPase were increased after training (113%, P < 0.05, 49%, P < 0.05, and 27%, P < 0.05, respectively). Whereas an acute bout of exercise increased the phosphorylation of PKCα/βII on Thr(638/641) pre- and posttraining, phosphorylation of PKCζ/λ on Thr(403/410) was increased in response to acute exercise only after the 10-day training. In conclusion, we show that only acute exercise, and not short-term training, increases phosphorylation of PLM on Ser(63) and Ser(68), and data from one-legged cycling indicate that this effect of exercise on PLM phosphorylation is not due to systemic factors. Our results provide evidence that phosphorylation of PLM may play a role in the acute regulation of the Na(+)-K(+)-ATPase response to exercise.