Introduction DNA methylation is an important epigenetic modification associated with transcriptional repression and plays key roles in normal cell growth as well as oncogenesis. Among the three main DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), DNMT3A mediates de novo DNA methylation. However, the general effect of DNMT3A on cell proliferation, metabolism, and downstream gene regulation is still to be unveiled.Method In this study, we successfully created DNMT3A-deficient HEK293 cells with frameshift mutations in the catalytic domain using CRISPR/Cas9 technology. The DNMT3A deficient cells showed a 21.5% reduction in global DNA methylation levels, leading to impaired cell proliferation as well as a blockage of MAPK and PI3K-Akt pathways in comparison with wild-type cells.Result RNA-seq analysis demonstrated that DNMT3A knockout resulted in the up-regulation of genes and pathways related to cell metabolism but down-regulation of those involved in ribosome function, potentially explaining the growth and signaling pathways inhibition. Furthermore, DNMT3A ablation reduced DNMT3B gene methylation, explaining the down-regulated profiles of genes.Conclusion Our findings suggest a complex epigenetic regulatory role for DNMT3A, and the compensatory upregulation of DNMT3B in response to DNMT3A deficiency warrants further investigation to be validated in future studies.