Although gender mainstreaming has been long recognized as a strategy for addressing gender inequalities and associated negative health outcomes; its implementation has remained a challenge, even in the area of prevention of mother to child transmission of HIV (PMTCT). Despite recognition of gender in Tanzania's political arena and prioritization of PMTCT by the health sector, there is very little information on how well gender has been mainstreamed into National PMTCT guidelines and organizational practices at service delivery level. Using a case study methodology, we combined document review with key informant interviews to assess gender mainstreaming in PMTCT on paper and in practice in Tanzania. We reviewed PMTCT policy/strategy documents using the WHO's Gender Responsive Assessment Scale (GRAS). The scale differentiates between level 1 (gender unequal), 2 (gender blind), 3 (gender sensitive), 4 (gender specific), and 5 (gender transformative). Key informant interviews were also conducted with 26 leaders purposively sampled from three government health facilities in Mwanza city to understand their practices. The gender responsiveness of PMTCT policy/strategy documents varies, with some being at GRAS level 3 (gender sensitive) and others at GRAS level 4 (gender specific). Those which are gender sensitive indicate gender awareness, but no remedial action is developed; while those which are gender specific go beyond indicating how gender may hinder PMTCT to highlighting remedial measures, such as the promotion of couple counselling and testing for HIV. In addition, interviews on organizational processes and practices suggested that there has been little attention to the holistic integration of gender in the delivery of PMTCT services. The study has revealed limited integration of gender concerns in PMTCT policy documents. Similarly, health facility leader responses indicate perspectives and practices that pay little attention to the holistic integration of gender in the delivery PMTCT services.