ObjectiveThis study was aimed to identify determinants of HIV infection among children born from mothers on the prevention of mother to child transmission (PMTCT) programme in Southern Ethiopia. It was designed to explore the main contributors to the considerable transmission rate of HIV from mother to child.Setting and designA multicentre facility-based unmatched case–control study was conducted using 27 health facilities providing PMTCT service in Southern Ethiopia.ParticipantsOut of 307 (62 cases and 245 controls) expected to participate in this study, a total of 290 mother–child pairs of 58 cases and 232 controls have completed the interview. Cases were children born to mothers on PMTCT programme and with DNA PCR or antibody HIV positive test result at ≤24 months of age. Controls were children born to mothers on PMTCT programme and with DNA PCR or antibody HIV negative test result at ≤24 months of age.ResultData were collected from the mother and record and analysed using SPSS V.20. Logistic regression analysis was done for statistical association and the significance of association was declared at a p value of <0.05. Rural residence (adjusted OR (AOR): 4.15, 95% CI: (1.57 to 10.97)), knowing serostatus during current pregnancy (AOR: 5.11, 95% CI: (1.33 to 19.69)), home delivery (AOR: 6.00, 95% CI: (2.310 to 15.593)), poor partner involvement (AOR: 5.95, 95% CI: 1.91 to 18.53)), poor adherence, late enrolment of the child for ARV prophylaxis (AOR: 4.89, 95% CI: 1.34 to 17.88)), mixed breastfeeding practice (AOR: 10.36, 95% CI: (3.10 to 34.60)) and failure to be on cotrimoxazole therapy (AOR: 7.56, 95% CI: 2.07 to 27.61)) were factors significantly associated with MTCT.ConclusionThe finding implies that more needs to be done on rural residents, strengthening screening for HIV before pregnancy, encouraging male involvement, early enrolment of child for ARV prophylaxis, avoiding mixed breast feeding and putting newborn on cotrimoxazole therapy.