To induce the development of tertiary lymphoid organs (TLO) in a mouse model of melanoma and to evaluate TLO's functions in antitumor immunity.Lymphotoxin-beta receptor (LTβR) was overexpressed in NIH3T3 cells through the lentivirus system and the overexpression efficiency of LTβR in LTβR-NIH3T3 cells was examined. Western blot and qPCR were used to examine the non-canonical nuclear factor (NF)-κB signaling pathway in NIH3T3 cells overexpressing LTβR. B16-OVA melanoma mouse model was constructed to explore the induction of TLO and anti-tumor functions of TLO in LTβR-NIH3T3 cells.LTβR was overexpressed in NIH3T3 cells through the lentivirus system, and flow cytometry showed that the proportion of GFP + cells reached 99%. The overexpression of LTβR activated the non-canonical NF-κB signaling pathway in NIH3T3 cells. Findings from the mouse tumor model suggest that the injection of LTβR-NIH3T3 cells successfully induced the development of lymphoid tissue around the tumor and enhanced the tumor infiltration of T cells and MHCⅡ + macrophages, significantly inhibiting tumor growth and prolonging the survival of tumor-bearing mice.LTβR-NIH3T3 cells promoted anti-tumor immunity by inducing TLO development, which may provide new perspectives for tumor immunotherapy.