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Aggressive B-cell lymphoma is one of the most common types of blood malignancy. Robust delivery of genes of interest into target cells, long-term gene expression, and minimal risk of secondary effects are highly desirable for translational medicine including gene therapy and studies on gene function. However, efficient gene delivery into viral or nonviral B-lymphoma cells remains a challenge. Here, we report a strategy for inducing foreign gene expression in B-lymphoma cells by using a vector based on the novel parainfluenza virus PIV5-L (a strain isolated from B cells) that enabled us to study and control the function of a gene product within B-lymphoma cells. Using enhanced green fluorescent protein (eGFP) as a reporter, we successfully rescued PIV5-L and established a one-step system to generate PIV5-L virus-like particles (L-VLPs) with efficient delivery into a broad spectrum of susceptible B-lymphoma cell lines, including Epstein-Barr virus (EBV)- or Kaposi’s sarcoma-associated herpesvirus (KSHV)-transformed B-lymphoblastoid cells. Similar to lentiviral vector, the L-VLP highly expressed exogenous genes and remained stable for long periods without obvious negative effects on cell viability. Taken together, these data demonstrate that the PIV5-L-based system provides a potential new strategy for the delivery of desirable genes and the treatment of cancer. IMPORTANCE B-cell lymphoma is a common aggressive neoplastic disorder of lymphocytes. Delivery of genes of interest into B cells, particularly virus-mediated B-lymphoma cells, is still a challenge. In this study, we report that a system (L-VLP) based on the parainfluenza virus PIV5-L strain isolated from B cells had highly expressed exogenous genes and remained stable without obvious cell toxicity, which provides a potential new strategy for gene delivery and treatment of B-cell cancer.