Objective To establish a method for the isolation and culture of primary Leydig cells in tree shrew, to explore the infection characteristics of Zika virus (Zikv) on these cells, and to provide a basis for research on the damage and sterility of testis caused by Zikv infection. Methods A 3-month-old male tree shrew was sacrificed with 0. 4 mL of 3% sodium pentobarbital anesthetized and the testis was collected. A single-cell suspension was obtained by combined digestion with collagenase IV and trypsin. After purification, cells were identified by fluorescent staining with 3β-hydroxysteroid dehydrogenase, and the ability of cells to secrete testosterone was detected by ELISA. Tree shrew Leydig cells were infected with Zikv, as were kidney cells from African green monkeys susceptible to Zika virus for comparison. The viral loads in the cells and their culture supernatants at different time points of infection were determined, cytopathic changes were observed under a microscope, and testosterone content was determined by ELISA. Results The positive rate of isolated and cultured Leydig cells identified by 3β-hydroxysteroid dehydrogenase was as high as 98%. They proliferated rapidly and continuously secreted testosterone. The forth-generation Leydig cells secreted 1. 253 and 1. 163 ng / mL testosterone at 0 ~ 24 h and 24~ 48 h, respectively. Upon infection of Leydig cells of tree shrew with Zikv, the virus copy number reached 2. 15×10⁵ copies/ mL within 6 h, the cells’ ability to secrete testosterone was basically lost after 48 h of infection, and cytopathic effects were seen on the third day of infection. Conclusions A method for isolating and culturing Leydig cells from tree shrew was successfully established. The findings showed that Zikv could infect Leydig cells of tree shrew and lead to the loss of their testosterone secretion function, providing a basis for research on the mechanism by which Zikv affects the reproductive system.