Abstract: Tenofovir disoproxil fumarate (TDF) is a nucleoside analogue that has been widely used for clinical treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infection. The aim of this study was to investigate whether TDF has anti-Zika virus (ZIKV) activity in vitro. The inhibitory effect of TDF on ZIKV was detected by plaque reduction assay. Then, the anti-ZIKV activity of TDF at RNA level and protein level was verified by real time quantitative PCR and Western blot. Finally, MTT assay was used to determine the cytotoxicity of TDF. Our results showed that TDF not only reduced the formation of plaque after ZIKV infection, but also inhibited the replication of ZIKV RNA or expression of ZIKV NS2B protein. The 50% effective concentration (EC₅₀) of TDF in inhibition of ZIKV replication were 14.96-27.47 μmol·L^-1, while that of ribavirin was 56.01 ±12.16 μmol·L^-1, which served as the positive control. The cytotoxicity of TDF and ribavirin in Vero cells were very low, with their 50% cytotoxic concentration (CC₅₀) values being greater than 500 μmol·L^-1. The therapeutic index of TDF calculated by CC₅₀/EC₅₀ was greater than 18.20, which was significantly higher than that of ribavirin. The results suggest that TDF has good anti-ZIKV activity in vitro and is expected to become a candidate drug for anti-ZIKV therapy.