Zika virus (ZIKV) is a mosquito-borne flavivirus, and its infection may cause severe neurodegenerative diseases. The outbreak of ZIKV in 2015 in South American has caused severe human congenital and neurologic disorders. Thus, it is vitally important to figure out inner mechanism of ZIKV infection. Here, our data suggested that the ubiquitin-specific peptidase 38 (USP38) played an important role in host resistance to ZIKV infection, during which ZIKV infection did not affect USP38 expression. Mechanistically, USP38 bound to ZIKV envelope (E) protein through its C-terminal domain and attenuated its K48-linked and K63-linked polyubiquitination, thereby repressed the infection of ZIKV. In addition, we found that the deubiquitinase activity of USP38 was essential to inhibit ZIKV infection, and the mutant that lacked the deubiquitinase activity of USP38 lost ability to inhibit the infection. In conclusion, we found a novel host protein USP38 against ZIKV infection, and this may represent a potential therapeutic target for the treatment and prevention of ZIKV infection.