Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function.