Our previous observations indicated that extractable organic matter (EOM) from PM2.5 induced malformations in the heart of zebrafish embryos by activating AhR and inhibiting canonical Wnt/β-catenin signal pathway. As a nutritional factor, folic acid (FA) is reported to prevent cardiac defects during embryo development. Hence, we hypothesize that FA may prevent PM2.5-induced heart defects by interfering with AhR and Wnt/β-catenin signaling pathways. Our results showed that FA supplementation alleviated the EOM-induced heart defects in zebrafish embryos, and both AhR inhibitor CH223191 and Wnt activator CHIR99021 enhanced the protective efficiency of FA. Furthermore, FA supplementation attenuated the EOM-induced upregulation of AhR and its target genes including Cyp1a1, Cyp1b1, Ahrra, and Ahrrb. EROD assay confirmed that the EOM agonized Cyp1a1 activity was diminished by FA. The EOM-induced downregulation of β-catenin and its target genes including Nkx2.5, Axin2, Sox9b, and Cox2b were recovered or even overexpressed in embryos exposed to EOM plus FA. In conclusion, our study suggested that FA supplementation protected against PM2.5 cardiac development toxicity by targeting AhR and Wnt/β-catenin signal pathways.