Decreasing muscle phosphagen content through dietary administration of the creatine analogue, -guanidinopropionic acid (-GPA), improves skeletal muscle oxidative capacity and resistance to fatigue during aerobic exercise in rodents, similar to that observed with endurance training. Surprisingly, the effect of -GPA on muscle substrate metabolism has been relatively unexamined, with only a few reports of increased muscle GLUT4 content and insulin-stimulated glucose uptake/clearance in rodent muscle. The effect of chronically decreasing muscle phophagen content on muscle FA metabolism (transport, oxidation, esterification) is virtually unknown. The purpose of the present study was to examine changes in muscle substrate metabolism in response to 8 wk feeding of -GPA. Consistent with other reports, -GPA feeding decreased muscle ATP and total creatine content by ~50 and 90%, respectively. This decline in energy charge was associated with simultaneous increases in both glucose (GLUT4; +33 to 45%, P<0.01) and FA (FAT/CD36; +28 to 33%, P<0.05) transporters in the sarcolemma of red and white muscle. Accordingly, we also observed significant increases in insulin-stimulated glucose transport (+47%, P<0.05) and AICAR-stimulated palmitate oxidation (+77%, P<0.01) in the soleus muscle of -GPA fed animals. Phosphorylation of AMPK (+20%, P<0.05), but not total protein, was significantly increased in both fibre types in response to muscle phosphagen reduction. Thus, the content of sarcolemmal transporters for both of the major energy substrates for muscle increased in response to a reduced energy charge. Increased phosphorylation of AMPK may be one of the triggers for this response.