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Parental vaccine hesitancy (VH) is a major barrier to childhood vaccination. We aimed to identify the determinants of parental VH towards routine childhood immunization in 12 Sub-Saharan African countries. A cross-sectional study was conducted from November 1 to December 15, 2022. Parents of children aged 19 months to 6 years and residing in the Sub-Saharan Africa were included. An anonymous online survey and face-to-face interviews were conducted. The Parent Attitude about Childhood Vaccine Scale was used to identify vaccine-hesitant parents. Multivariate regression and mediating analysis were performed. Across the 5032 participants, 21.2% were hesitant towards routine childhood immunization. Urban residents (adjusted odds ratio [AOR] = 1.32, 95% confidence interval [CI]: 1.10–1.58), non-first-born children (AOR = 1.54, 95% CI: 1.19–1.98), and chronically ill children (AOR = 2.00, 95% CI: 1.69–2.37) increased the likelihood of parental VH. Mothers with higher education, attending at least one antenatal care (ANC) visit (AOR = 0.25, 95% CI: 0.19–0.32), and had a healthcare facility–based delivery (AOR = 0.55, 95% CI: 0.44–0.70) decreased the odds of parental VH. Parental VH mediated the effect of ANC and mothers' age on vaccination uptake. ANC increased the odds of vaccination uptake (odds ratio [OR] = 12.49, 95% CI: 9.68–16.13). Parental VH mediated the association between ANC and vaccination uptake, decreasing the likelihood of vaccination uptake (OR = 0.12, 95% CI: 0.10–0.14). Each additional year of the mother's age decreased the odds of vaccination uptake (OR = 0.95, 95% CI: 0.95–0.96). The indirect effect of mother's age on vaccination through parental VH decreased the odds of vaccination uptake (OR = 0.45, 95% CI: 0.44–0.45). Parental VH continued to be a mediator of the combined effect of mother's age and ANC on vaccination uptake, decreasing the likelihood of vaccination uptake (OR = 0.0017, 95% CI: 0.00166–0.00168). Context-specific interventions are needed to address parental VH and improve vaccine acceptance and coverage. [ABSTRACT FROM AUTHOR]