[Abstract] Objective: To investigate the effect of IL-27 in combination with IL-15 on the anti-tumor effects of NK92 cells and the possible molecular and signaling mechanisms. Methods: NK92 cells with high IL-15 expression (IL-15-NK92 cells) were cultured in different mass concentrations of IL-27 (0, 10, 20, 30 and 60 ng/ml) for 24 h. The effects of IL-27 on IL-15 secretion, migration and proliferation of IL-15-NK92 cells were detected by ELISA, Transwell and CCK-8 assay, respectively. Flow cytometry was used to detect the expression levels of IL-15-NK92 cell surface receptors NKG2D, NKp30 and NKp46, as well as the secretion levels of perforin and granzyme B. LDH method was used to detect the cytotoxic effect of IL-15-NK92 cells on hematologic tumor cells and solid tumor cells, and WB was used to detect the expressions and phosphorylation level of STATs pathway-related proteins. Results: IL-27 at the concentration of 30 ng/ml promoted IL-15-NK92 cells secreting IL-15 (P<0.01), significantly enhanced the cell migration (P<0.05) but inhibited the proliferation of IL-15-NK92 cells (P<0.05). 30 ng/ml IL-27 could significantly promote the expressions of NKG2D, NKp30 and NKp46 on surface of IL-15-NK 92 cells, as well as elevate the secretion of perforin (all P<0.05), but didn’t affect the secretion of granzyme B (P>0.05); moreover, it also significantly enhanced the cytotoxicity of IL-15-NK92 cells against hematologic malignancies and solid tumor cells (P<0.05 or P<0.01), and up-regulated the phosphorylation levels of STAT1, STAT3 and STAT5 (all P<0.01). Conclusion: IL-27 can enhance the cytotoxicity of IL-15-NK92 cells against hematologic tumor cells and solid tumor cells, which might be related with its upregulation of phosphorylation level of STAT1, STAT3 and STAT5 in JAK-STAT pathway and multiple activating receptors in IL-15-NK92 cells.