Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii that affects reproductive performance in small ruminants. Although the T. gondii life cycle is well understood since 1960s, several aspects related to its infection remain unclear. In the present study we hypothesized that sheep inseminated with T. gondii-contaminated semen would develop toxoplasmosis. In order to test that hypothesis, 41 sheep were experimentally infected with semen spiked with the organism. Females were divided in three groups (G1-G3): (a) females in G1 group were inseminated with semen containing 6.5 x 10(4) tachyzoites; (b) females in G2 group with semen containing 4 x 10(7) tachyzoites; and (c) females in G3 group with tachyzoite-free semen (control group). To confirm T. gondii infection via semen, serological tests were performed using indirect immunofluorescence reaction and the detection of parasite DNA in the blood stream using the nested PCR test. While in G1 group only 5/15 (33.3%) of the females presented seroconversion, all sheep in G2 15/15 (100%) seroconverted. The nested PCR test showed that 14/15 (93.3%) of the females in the G1 and 14/15 (93.3%) in the G2 group were positive for T. gondii while in the G3 group all samples were negative. In addition, ultra-sound test evidenced that in sheep presented embryonic reabsorption in animals from the infected groups. In conclusion, insemination using fresh semen experimentally contaminated with different infectant doses of T. gondii tachyzoites was able to infect sheep, leading to the possibility of toxoplasmosis transmission via semen.