Zika virus (ZIKV) is a mosquito borne flavivirus originally confined to Africa and Asia that has spread to islands located in Southeast Asia, and most recently to the Americas and the Caribbean. Approximately 80% of infected individuals are asymptomatic, while the remaining infected population exhibit mild febrile syndrome such as rash, conjunctivitis, and arthralgia. In some adults, ZIKV causes neurotropic Guillain Barré syndrome (1). Vertical transmission of ZIKV in infected mothers causes fetal growth restriction, microcephaly, and congenital eye disease (2-5). Cases of ZIKV sexual transmission from male to female (6-8), male to male (9), and a suspected case of female to male transmission (10) have been reported. ZIKV has been detected in the semen of infected males (11-15), even after months of symptom onset (16-19). Viral persistence in the testes and semen can increase the risk of ZIKV transmission through rectal route in men having sex with men (MSM) and between heterosexual partners. The anorectal mucosa is a major entry site for HIV-1 transmission among MSM (20), and for the acquisition and transmission of other sexually transmitted diseases, such as syphilis, chlamydia, and gonorrhea. Although the risk of ZIKV acquisition through the rectal route is high, no pathobiological information is available. Here, we describe the establishment of a rectal route of ZIKV infection system using immunocompromised (ifnar1 -/-) male mice to determine their susceptibility to ZIKV and to assess viral dissemination to male reproductive organs. We found that rectal inoculation of ZIKV results in viremia with non-lethal infection. The rectal mucosa is susceptible to ZIKV entry and replication. Following rectal inoculation, ZIKV establishes active testicular infection that persists at least 21 days. During the acute phase of infection, the highest viral load was observed in the spleen, with inflammatory and immune cellular infiltration. Macrophages in the splenic red pulp are the target cells for ZIKV infection.