Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of aggressive B-cell non-Hodgkin lymphoma. About one-third of patients are either refractory to the treatment or experience relapse afterwards, pointing to the necessity of developing other effective therapies for DLBCL. Human B-lymphocytes are susceptible to JC polyomavirus (JCPyV) infection, and JCPyV virus-like particles (VLPs) can effectively deliver exogenous genes to susceptible cells for expression, suggesting the feasibility of using JCPyV VLPs as gene therapy vectors for DLBCL. The JCPyV VLPs packaged with a GFP reporter gene were used to infect human DLBCL cells for gene delivery assay. Furthermore, we packaged JCPyV VLPs with a suicide gene encoding thymidine kinase (TK) to inhibit the growth of DLBCL in vitro and in vivo. Here, we show that JCPyV VLPs effectively entered human germinal center B-cell-like (GCB-like) DLBCL and activated B-cell-like (ABC-like) DLBCL and expressed the packaged reporter gene in vitro. As measured by the MTT assay, treatment with tk-VLPs in combination with gancyclovir (GCV) reduced the viability of DLBCL cells by 60%. In the xenograft mouse model, injection of tk-VLPs through the tail vein in combination with GCV administration resulted in a potent 80% inhibition of DLBCL tumor nodule growth. Our results demonstrate the effectiveness of JCPyV VLPs as gene therapy vectors for human DLBCL and provide a potential new strategy for the treatment of DLBCL.