Abstract: Objective To investigate the mechanism by which folic acid prevents neural tube defects (NTDs). Methods PC12 cells were randomly divided into three groups: a control group, a model group, and a folic acid group. A NTD cell injury model was established by treating PC12 cells with all-trans retinoic acid (atRA). PC12 cell viability was evaluated by CCK-8 assay. The apoptotic rate was detected by flow cytometry. The total cellular RNA was sequenced by RNA sequencing (RNA-seq) technology to screen for differentially expressed genes (DEGs). Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the DEG set to identify apoptosis-related genes. RT-qPCR was used to detect the changes in the expression of apoptosis-related genes, including caspase-4, caspase-12, Ern1, and CD44. Results Compared with the control group, PC12 cell viability in the model group significantly decreased (P<0.05), while the apoptotic rate significantly increased (P<0.01). Compared with the model group, the folic acid group showed significantly increased cell viability (P<0.05) and significantly reduced apoptotic rate (P<0.01). GO functional analysis of the DEG set revealed significant enrichment in endoplasmic reticulum stress-mediated apoptosis-related GO terms, such as "intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress". KEGG pathway analysis indicated significant enrichment in pathways such as "cell cycle" and "DNA replication". Compared with the control group, the model group showed upregulation of apoptosis-related genes caspase-12, caspase-4, Ern1, and CD44. Compared with the model group, the folic acid group exhibited downregulation of these apoptosis-related genes. RT-qPCR results showed that, compared with the control group, the model group showed significant increases in the mRNA levels of caspase-12, caspase-4, Ern1, and CD44 (P<0.05 and P<0.01), while the folic acid group presented significantly lower mRNA levels of caspase-12, caspase-4, Ern1, and CD44 than the model group (P<0.05 and P<0.01). Conclusions Folic acid inhibits atRA-induced apoptosis in PC12 cells, and protects neuron-like cells. This mechanism may be related to the downregulation of apoptosis-related genes caspase-12, caspase-4, Ern1, and CD44, suggesting that folic acid exerts its protective effects by inhibiting endoplasmic reticulum stress-mediated apoptosis.