Cathelicidins have been shown to effectively inhibit flavivirus replication in vitro. However, the effects of mouse and human endogenous cathelicidins on flavivirus infection in vivo are rarely known. We herein found that mouse endogenous cathelicidin CRAMP was significantly up-regulated upon Zika virus (ZIKV) infection. CRAMP deficiency markedly exacerbated ZIKV replication in testis, and aggravated ZIKV-induced testicular damage and spermatic damage in mice, indicating that endogenous cathelicidin is required for protection against ZIKV-caused male infertility in mice. In vitro antiviral assay showed that both mouse cathelidin CRAMP and human cathelicidin LL-37 obviously reduced ZIKV-caused cytopathic effect and inhibited ZIKV replication in Vero cells. Antiviral mechanism revealed that they both directly inactivated ZIKV virons by binding to ZIKV virons and inducing the leakage of ZIKV genomic RNA, consequently inactivated ZIKV virons. In vivo antiviral assay indicated that both of them effectively inhibited ZIKV replication in C57BL/6J and IFNα/β receptor-deficient (Ifnar1) mice when CRAMP or LL-37 was intravenously injected in parallel with or at 1 h after intravenous injection of ZIKV, implying that CRAMP and LL-37 effectively inactivated ZIKV particles and exhibited therapeutic potential against ZIKV infection in vivo. Our findings reveal that endogenous cathelicidin CRAMP and LL-37 act as inactivators of ZIKV, and effectively protect against ZIKV replication and ZIKV-induced male infertility, highlighting their potential for therapy of ZIKV infection.