BACKGROUND: Despite more efforts for prevention of mother to child HIV transmission, still there are problems with provider-initiated HIV testing. This study was done to assess the acceptance rate of provider-initiated HIV testing among antenatal care attendants and its associated factors. METHODS: Institutions based cross sectional study with a sample size of 398 was conducted from February to March 2014 in two health facilities in Assosa town. Proportional allocation of the sample size of health facilities followed by systematic sampling method was done; data were collected using an interviewer administered questionnaire. Bivariate and multivariate regression analysis was employed using SPSS version 20. RESULTS: A total of 386 pregnant women participated with response rate 97 % and 312 (80.8 %) of them accepted provider-initiated HIV testing. The odds of acceptance of provider-initiated HIV testing was higher among rural residents (AOR 4.04; 95 % CI 1.24-13.11) than urban. It was also higher among students (AOR 6.00; 95 % CI 1.45-24.75), merchants (AOR 4.43; 95 % CI 1.18-16.68) and employed women (AOR 2.15; 95 % CI 1.08-4.30) than housewives. Pregnant women who had no stigmatized attitude towards people living with HIV/AIDS were more likely to accept testing (AOR 3.54; 95 % CI 1.23-10.16) than who had a strong stigmatized attitude. In addition, those who planned to disclose their test results from their husbands were higher odd of acceptance (AOR 14.85; 95 % CI 4.60-47.94) than who secreted. CONCLUSION: Acceptance of provider-initiated HIV testing among pregnant women attending for antenatal care services was relatively high. Mothers from urban residence, occupational satus being housewives, stigmatization and not having a plan to disclose the status of test results were negatively affect the acceptance of provider-initiated HIV testing. During counselling sessions, antenatal care providers should focus on barriers of provider-initiated HIV testing such as residence, occupational status, stigmatized attitudes and disclosure status of results of HIV tests.