BACKGROUND:Systematic reviews of prenatal alcohol exposure effects generally only include conventional observational studies. However, estimates from such studies are prone to confounding and other biases. OBJECTIVES:To systematically review the evidence on the effects of prenatal alcohol exposure from randomized controlled trials (RCTs) and observational designs using alternative analytical approaches to improve causal inference. SEARCH STRATEGY:Medline, Embase, Web of Science, PsychINFO from inception to 21 June 2018. Manual searches of reference lists of retrieved papers. SELECTION CRITERIA:RCTs of interventions to stop/reduce drinking in pregnancy and observational studies using alternative analytical methods (quasi-experimental studies e.g. Mendelian randomization and natural experiments, negative control comparisons) to determine the causal effects of prenatal alcohol exposure on pregnancy and longer-term offspring outcomes in human studies. DATA COLLECTION AND ANALYSIS:One reviewer extracted data and another checked extracted data. Risk of bias was assessed using customized risk of bias tools. A narrative synthesis of findings was carried out and a meta-analysis for one outcome. MAIN RESULTS:Twenty-three studies were included, representing five types of study design, including 1 RCT, 9 Mendelian randomization and 7 natural experiment studies, and reporting on over 30 outcomes. One study design-outcome combination included enough independent results to meta-analyse. Based on evidence from several studies, we found a likely causal detrimental role of prenatal alcohol exposure on cognitive outcomes, and weaker evidence for a role in low birthweight. CONCLUSION:None of the included studies was judged to be at low risk of bias in all domains, results should therefore be interpreted with caution. SYSTEMATIC REVIEW REGISTRATION:This study is registered with PROSPERO, registration number CRD42015015941.