To elucidate the functional significance of heat-shock protein beta 1 (HSPB1) in androgen-mediated myogenesis of bovine cells, we conducted 'loss and gain of function of HSPB1' assays by siRNA inhibition and gene overexpression. siRNA inhibition of HSPB1 expression reduced the expression of desmin (a myogenic marker) and repressed the formation of myotubes in cells induced for myogenic differentiation. In contrast, overexpression of HSPB1 enhanced the expression of desmin and accelerated formation of myotubes. The loss and gain of HSPB1 function was closely associated with the expression level of androgen receptor (AR). Our findings suggest that HSPB1 mediates androgen signaling by binding directly to AR and then enhancing androgen-mediated myogenesis in myogenic cells.