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Zika virus (ZIKV) RNA has been found to remain in human semen for up to one year after infection, but the presence of Flavivirus antigens in the different compartments of semen has been largely unexplored. Following the introduction of ZIKV in Nicaragua (2016), a prospective study of patients with clinical symptoms consistent with ZIKV was conducted in Le&oacute, n to investigate virus shedding in different fluids. ZIKV infection was confirmed in 16 male subjects (&ge, 18 years of age) by RT-qPCR in either blood, saliva or urine. Of these, three provided semen samples at 7, 14, 21, 28, 60 and 180 days postsymptom onset (DPSO) for Flavivirus antigens and RNA studies. These cases were compared with 19 asymptomatic controls. Flavivirus antigens were examined by immunofluorescence (IF) using the 4G2 Mabs, and confocal microscopy was used to explore fluorescence patterns. The three (100%) symptomatic subjects and 3 (16%) of the 19 asymptomatic subjects had Flavivirus antigens and viral RNA in the spermatozoa fraction. The percentage of IF Flavivirus-positive spermatozoa cells ranged from 1.9% to 25% in specimens from symptomatic subjects, as compared with 0.8% to 3.8% in specimens from asymptomatic controls. A marked IF-pattern in the cytoplasmic droplets and tail of the spermatozoa was observed. The sperm concentrations (45 &times, 106/mL vs. 63.5 &times, 106/mL, p = 0.041) and the total motility percentage (54% vs. 75%, p = 0.009) were significantly lower in specimens from ZIKV-positive than in those of ZIKV-negative. In conclusion, this study demonstrated the presence of Flavivirus antigens and RNA within a time frame of 28 DPSO in sperm cells of symptomatic and asymptomatic subjects during the ZIKV epidemic. These findings have implications for public health, in terms of nonarthropod-born, silent transmission facilitated by sperm cells and potential transmission from asymptomatic males to pregnant women, with consequences to the fetus.