Introduction: Despite more efforts for prevention of mother to child HIV transmission, still there are problems with provider-initiated HIV testing at the health facilities. Objective: To assess the acceptability of provider-initiated HIV testing as an intervention for prevention of mother to child transmission of HIV among antenatal care attendants and its associated factors. Methods: Institution based cross sectional study with sample size of 398 was conducted from February to March 2014 in two health facilities of Assosa town. Proportional allocation of the sample size to health facilities followed by systematic sampling method was done; data were collected using interviewer administered questionnaire. Bivariate and multivariate regression analysis was employed using SPSS version 20. Result: A total of 386 pregnant women participated with response rate 97% and 312(80.8%) of them accepted provider-initiated HIV testing. The odd 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 high 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 and recommendation: Acceptance of provider-initiated HIV testing for antenatal care was relatively high among pregnant women. Stigmatization and disclose of results were negative effect for acceptance. During counselling sessions, antenatal care providers should focus on barriers of acceptability of HIV testing such as stigma and disclosed test results for increasing the acceptance rate of provider-initiated HIV testing.