This study aimed to investigate the effect of tryptophan on cell migration and its underlying mechanism in porcine intestine epithelial cells (IPEC-J2). This study shows that tryptophan can modulate IPEC-J2 cell proliferation, enhance cell migration and the protein concentration of calcium-sensing receptors (CaSR), total ras-related C3 botulinum toxin substrate 1 (total Rac1), Rho family member 1 of GTP-binding protein (GTP-rac1), and phosphorylated phospholipase Cγ1 (p-PLC-γ1). Moreover, Rac1, phospholipase C-γ1 (PLC-γ1) silencing or CaSR inhibitor (NPS2143) inhibited tryptophan-induced upregulation of cell migration. In contrast, tryptophan enhanced the cell migration area and protein concentration of total Rac1, GTP-rac1, and phosphorylated PLCγ1 in cells transfected with wild type CaSR. The overexpression of CaSR increased cell migration, which was reduced by Rac1 or PLC-γ1 silencing. Collectively, our results suggested that tryptophan can improve IPEC-J2 cell migration through the CaSR/Rac1/PLC-γ1 signaling pathway.