The phosphodiesterases inhibitor pentoxifylline gained attention for Duchenne muscular dystrophy therapy for its claimed anti-inflammatory, anti-oxidant and anti-fibrotic action. A recent finding also showed that pentoxifylline counteracts the abnormal over-activity of a voltage-independent calcium channel in myofibers of dystrophic mdx mice. The possible link between work-load, altered calcium homeostasis and oxidative stress, pushed toward a more detailed investigation. Thus, a 4-8 week treatment with pentoxifylline (50 mg/kg/day i.p.) was performed in mdx mice, undergoing or not a chronic exercise on treadmill. In vivo, the treatment partially increased forelimb strength and enhanced resistance to treadmill running in exercised animals. Ex vivo, pentoxifylline restored the mechanical threshold, an electrophysiological index of calcium homeostasis, and reduced resting cytosolic calcium in extensor digitorum longus muscle fibers. Mn-quenching and patch-clamp technique confirmed that this effect was paralleled by a drug-induced reduction of membrane permeability to calcium. The treatment also significantly enhanced isometric tetanic tension in mdx diaphragm. The plasma levels of creatine kinase and reactive oxygen species (ROS) were both significantly reduced in treated-exercised animals. Dihydroethidium staining, used as an indicator of ROS production, showed that pentoxifylline significantly reduced the exercise-induced increase in fluorescence in the mdx tibialis anterior muscle (TA). A significant decrease in connective tissue area and pro-fibrotic cytokine TGF-1 was solely found in TA muscle. In both diaphragm and gastrocnemious muscle a significant increase in NCAM positive area was instead observed. This data supports the interest toward pentoxifylline and allows insight in the level of cross-talk between pathogenetic events in work-loaded dystrophic muscle.