Skeletal muscle has emerged as an endocrine organ secreting exercise-induced factors (exerkines), which play a pivotal role in inter-organ crosstalk. Via MS-based proteomics, we identified thymosin beta-4 (TMSB4X) to be the most upregulated secreted protein in the media of contracting C2C12 myotubes. TMSB4X was also acutely increased in the plasma of exercising humans and there was a net uptake of TMSB4X across the thigh during exercise. Chronic treatment of TMSB4X in mice did not ameliorate the metabolic disruptions associated with diet induced-obesity, nor did it enhance muscle regeneration in vivo. However, TMSB4X did increase osteoblast proliferation and neurite outgrowth, consistent with its WADA-classification as a prohibited growth factor. Thus, we report TMSB4X as a novel human exerkine with a potential role in cellular crosstalk.