Schwann cells migrate along axons before initiating myelination during development and their migration facilitates peripheral nerve regeneration after injury. Axon guidance molecule Slit‐2 is highly expressed during peripheral development and nerve regeneration; however, whether Slit‐2 regulates the migration of Schwann cells remains a mystery. Here we show that Slit‐2 receptor Robo‐1 and Robo‐2 were highly expressed in Schwann cells in vitro and in vivo. Using three distinct migration assays, we found that Slit‐2 repelled the migration of cultured Schwann cells. Furthermore, frontal application of a Slit‐2 gradient to migrating Schwann cells first caused the collapse of leading front, and then reversed soma translocation of Schwann cells. The repulsive effects of Slit‐2 on Schwann cell migration depended on a Ca²⁺ signaling release from internal stores. Interestingly, in response to Slit‐2 stimulation, the collapse of leading front required the loss of F‐actin and focal adhesion, whereas the subsequent reversal of soma translocation depended on RhoA‐Rock‐Myosin signaling pathways. Taken together, we demonstrate that Slit‐2 repels the migration of cultured Schwann cells through RhoA‐Myosin signaling pathways in a Ca²⁺‐dependent manner.