Thymosin β4 (Tβ4 ) is a regenerative peptide that we hypothesized would promote healing of fractured bone. Mice received a bilateral fibular osteotomy and were given i.p. injections of either Tβ4 (6 mg/kg) or saline. Calluses from saline- and Tβ4 -treated mice were analyzed for: (1) biomechanical properties and (2) composition using micro-computed tomography (µCT) and histomorphometry. Biomechanical analysis showed that Tβ4 -treated calluses had a 41% increase in peak force to failure (p < 0.01) and were approximately 25% stiffer (p < 0.05) than saline-treated controls. µCT analysis at 21 days post-fracture showed that the fractional volume of new mineralized tissue and new highly mineralized tissue were respectively 18% and 26% greater in calluses from Tβ4 -treated mice compared to controls (p < 0.01; p < 0.05, respectively). Histomorphometry complemented the µCT data; at 21 days post-fracture, Tβ4 -treated calluses were almost 23% smaller (p < 0.05), had nearly 47% less old cortical bone (p < 0.05) and had a 31% increase in new trabecular bone area/total callus area fraction compared with controls (p < 0.05). Our finding of enhanced biomechanical properties of fractures in mice treated with Tβ4 provides novel evidence of the therapeutic potential of this peptide for treating bone fractures.