Maternal alcohol consumption during pregnancy can induce central nervous system abnormalities in the fetus, and folic acid supplementation can reverse some of the effects. The objective of the present study was to investigate prenatal alcohol exposure-induced fetal brain proteome alteration and the protective effect of folic acid using proteomic techniques. Alcohol (5·0 g/kg) was given intragastrically from gestational day (GD) 6 to15, with or without 60·0 mg folic acid/kg given intragastrically during GD1–16 to pregnant Balb/c mice. The control group received distilled water only. Results of litter evaluation on GD18 showed that supplementation of folic acid reversed the prevalence of microcephaly induced by alcohol. Proteomic analysis indicated that, under the dosage of the present investigation, folic acid mainly reversed the alcohol-altered proteins involved in energy production, signal pathways and protein translation, which are all important for central nervous system development.