Background: Prevention of mother to child transmission of Human Immunodeficiency Virus (PMTCT) program requires great male participation to attain Ethiopia' s new plan to elimination of mother to child transmission of Human Immunodeficiency Virus. In this regard, antenatal care is one of the areas where male couple is expected to participate through voluntary counseling and testing Objectives: To assesses partner testing in prevention of mother to child transmission of HIV and associated factors at antenatal care in woreta town, south Gondar zone, and Amahara region. Methods: A facility-based cross-sectional study was conducted. A total of 422 male partners, whose spouses were attending antenatal care at Woreta health center were enrolled through systematic sampling method. The study was also supplemented by focus group discussions. The data were cleaned and coded then analyzed using SPSS for windows version 20. Odds ratio were computed to assess the presence and degree of association between dependent and independent variables. Logistic regression analysis was carried out to see independent effect of each variable on the outcome. Qualitative data was also analyzed by thematic content. Result: Only 33.4% of male partners accompanied their spouses for HIV testing and counseling at antenatal clinic. Men having no formal education was 90% less likely to be tested as compared to those who had completed the higher education [AOR=O.l, 95%CI: 0.023-0.050, P=0.004]. Identified barriers to partner testing in the PMTCT program were individual factors such as being busy, proxy testing, fear of being tested and neglecting importance, inadequate knowledge about the. program, health system factors and absence of legally enforced law that makes male to participate. Conclusion and Recommendation: Majority of male partners had not participated in prevention of mother to child transmission (PMTCT) program at antenatal clinic. To promote uptake of the service by male partner, there is a need to raise awareness of the program among community through behavioral change and communication, develop a new strategy for promoting male participation in the program and couple HIV testing laws should be revised to get HIV free generation.