This study investigated the in vitro growth characteristics and differential potential of muscle-derived satellite cells (MDSCs) derived from rats at different postnatal (P) stages, in order to expand the range of source material for tissue engineering. Rat MDSCs were isolated from P5, P10, P15, P21 and P42 rat skeletal muscles using double enzyme digestion and differential adherent culture. Neurogenic, osteogenic and myogenic induction media were used to induce directed differentiation. Differentiated nerve cells, osteoblasts and myotubes were identified by their morphology and immunohistochemical staining. Most cells transformed into spindle-shaped mononuclear cells after 48 h and proliferated rapidly. MDSCs were difficult to isolate from P42 rats. After neurogenesis, four groups MDSCs formed neuron-specific enolase positive polygonal-shaped dendritic cells. After osteogenesis, P5, P10, P15 and P21 MDSCs formed Alizarin red- and osteocalcin-positive bone nodules. After myogenesis, myotubes were formed and were fast muscle myosin-positive. MDSCs derived from P5, P10, P15 and P21 rat skeletal muscle are easy to isolate, culture and amplify in vitro, which increases the range of source material available for tissue engineering.