Although tumor cell lysate (TCL) is a type of immunocyte stimulator, its immunosuppressive function must not be ignored. The present study reported that TCL prepared from a Lewis lung cancer cell was able to induce the development of immunosuppressive macrophages (MΦ) and tolerogenic dendritic cells. In addition, TCL upregulated the expression of CD69 in mouse splenocytes, and cell apoptosis and the percentage of regulatory T cells in mouse splenocytes simultaneously increased. Furthermore, the present study found that the immunosuppressive factor, hyaluronan, and the apoptosis inducers, Fas ligand and transforming growth factor-β, are present in TCL. These components may be associated with the emergence of immunosuppressive cells or splenocyte apoptosis. Thus, the present study has enriched our understanding of the composition of TCL and its negative regulatory effect on immunocytes.