Atnafu Gebeyehu N, et al. - Given that for terminating mother to child transmission of the virus among pregnant women, acceptance of human immune virus testing is an entry point of intervention, researchers conducted an institutional-based cross-sectional study with a sample size of 340 from May 20 to June 15, 2019, to assess acceptance of HIV testing and associated factors among these pregnant women who attended antenatal care in Gunino Health Center, Southern Ethiopia 2019. Among the total of 340 pregnant women who participated with a response rate of 96%, testing was accepted by 234 (68.8%). This suggests a relatively low acceptance of human immune virus testing among pregnant women attending antenatal care clinics. Respondents who had awareness about mother to child transmission had higher odds of acceptance of human immune virus testing than their counterparts. In addition, it was higher among respondents who had understood the benefit of testing than those who did not recognize the benefit of testing. A higher likelihood to accept testing was noted among participants who were knowledgeable about the prevention of mother to child transmission than their counterparts. Based on these findings, they recommend antenatal caregivers to emphasis decreasing barriers to testing, like lack of awareness, knowledge, and educational background.