Cathelicidins (CATHs) are a family of endogenous antimicrobial peptides that are capable of both direct bacteria-killing and immunomodulatory effects. P2X7 receptor (P2X7R) is a mediator of CATH in mammalian immune cells. Here, we studied the function and regulation of CATH in head kidney-derived monocytes/macrophages (MO/MΦ) from ayu, Plecoglossus altivelis. We investigated the chemotaxis of MO/MΦ in response to ayu CATH (PaCATH), and found that PaCATH had a dose-dependent effect on MO/MΦ chemotaxis with the optimal concentration of 10.0 μg/ml. The qPCR and Western blot analysis revealed that PaCATH inhibited the expression of ayu P2X7R (PaP2X7R) at both mRNA and protein levels. Knockdown of the PaP2X7R expression in ayu MO/MΦ by RNA interference not only significantly inhibited the chemotactic effect of PaCATH on MO/MΦ, but also obviously reduced the effect of PaCATH on the phagocytosis, bacteria-killing, respiratory burst, and cytokine expression of ayu MO/MΦ. Our study revealed that the immunomodulatory effect of fish CATH is mediated by P2X7R.