Background: The male partner's reaction is an important determinant in the uptake of prevention of mother–to–child transmission of HIV (PMTCT) service. Without knowing the determinant factors of male partner's reaction on PMTCT programmes, change will be very difficult or impossible to implement. Therefore, this study was conducted to identify the determinant factors of male partner's influence on pregnant women towards the PMTCT programme in Gondar town, North West Ethiopia. Methods: A cross-sectional quantitative study based in a health institution was conducted in Gondar town from 22 July–18 August 2011. A total of 410 pregnant women attending antenatal care (ANC) were recruited to participate using stratified sampling technique. Data were collected using a structured interviewer administered questionnaire. Multiple logistic regression analysis was employed using SPSS version 16. Results: Of the 400 pregnant women who actively participated in this study, 314 (78.5%) expected a negative reaction for HIV positive test result from their partners. A positive reaction from the partner was associated with women having their own income (Adjusted odds ratio (AOR) (95% CI)=2.180(1.213, 3.920)), residing in the urban areas (AOR (95%CI)=2.259(1.210, 4.217)), having education level of secondary level and above (AOR (95%CI)=6.048(3.121, 11.720)), not having a stigmatising attitude towards people living with HIV (AOR (95%CI)=2.149 (1.240, 3.725)), having a positive attitude towards counsellors (AOR (95%CI)=2.455(1.419, 4.246)) and being able to access health facilities (AOR (95%CI)=2.345(1.223, 4.498)). Conclusions: Most of the participants in this study expected their partner to react negatively towards a positive HIV test result. Since having their own income is strongly associated with a positive partner's reaction to PMTCT services, emphasis should be given for education and economic empowerment of women. A well functioning and accessible health facility with PMTCT service is important, especially in rural areas.