As humans age, they lose both muscle mass and strength (sarcopenia). Testosterone, a circulating hormone, progressively declines in aging and is associated with loss of muscle mass and strength. The surgical joining of a young and old mouse (heterochronic parabiosis) activates Notch signaling and restores muscle regenerative potential in aged mice. We hypothesize that testosterone is at least one of the factors required for the improvement seen in muscles in old mice in heterochronic parabiosis with young mice. To test this hypothesis, we established the following heterochronic parabioses between young (Y; 5 months old) and old (O; 22-23 months old) C57BL6 male mice: (1) Y:O; (2) castrated Y:O (ØY:O); (3) castrated + testosterone-treated Y:O (ØY + T:O). A group of normal young mice received empty implants, and old mice were used as controls. Parabiotic pairings were maintained for 4 weeks prior to analysis. Serum testosterone levels were three-fold higher in young than in old mice. The ØY + T:O pairing demonstrated significantly elevated levels of serum testosterone and an improvement in gastrocnemius muscle weight, muscle ultrastructure, muscle fiber cross-sectional area, and Notch-1 expression in old mice. These changes were not present in aged mice in the ØY:O pairing. These data indicate that testosterone has a critical role in mediating the improved muscle mass and ultrastructure seen in an experimental model of heterochronic parabiosis.