<p>A significant proportion of patients with recurrent and refractory diffuse large B-cell lymphoma (DLBCL) exhibit high levels of BCL-2 expression. However, some of these patients are resistant to BCL-2 inhibitors, and the underlying mechanisms remain unclear. In venetoclax-resistant DLBCL cell line, a Cell Counting Kit-8 (CCK8) assay was used to determine the half-maximal inhibitory concentration (IC₅₀) value. Flow cytometric was performed to determine mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and cell apoptosis rates, respectively. The construction of NSUN4 knockdown/overexpression cell line using CRISPR-Cas9 technology. KEGG pathway enrichment analysis indicated that NSUN4 modulates venetoclax resistance. Furthermore, we used Western blotting to explore the mechanism underlying venetoclax-resistant. Finally, the antileukemic activity was further evaluated in an in vivo xenograft model. We identified that NSUN4, an m5C methyltransferase, is highly expressed not only in the venetoclax-resistant cell line but also in the lymph nodes of recurrent and refractory DLBCL patients primarily by inhibiting the p53 signaling pathway, although the precise mechanism warrants further investigation, and is correlated with poor prognosis. Moreover, we discovered that Apatinib could reduce NSUN4 expression and effectively reverse venetoclax resistance. These findings suggest that NSUN4 is a critical target for overcoming venetoclax resistance in DLBCL patients. Our study reveals the role of NSUN4 and the p53 signaling pathway in venetoclax resistance at the molecular, cellular, and animal levels. Understanding how m5C methylation mediates venetoclax resistance and regulates the p53 pathway will provide a theoretical foundation for overcoming venetoclax resistance in patients with recurrent and refractory DLBCL.</p>