To investigate the effect and mechanism of inhibiting the activation of nucleotide binding oligomerization domain protein 2 (NOD2) on inflammatory response of hepatocellular carcinoma cells.Methods Hepatocellular carcinoma cells were cultured.Western blot was used to detect NOD2 expression in hepatocellular carcinoma cells and normal hepatocytes.Small interfering RNA (siRNA) was used to establish NOD2 knockdown agents (siNOD2 group) to inhibit NOD2 expression, while the negative control group was siNC group, and hepatocellular carcinoma cells were treated.Cell proliferation rate was measured by CCK-8 method and apoptosis rate was measured by flow cytometry.The expressions of NF-κB P65, STAT3 and NOD2 were detected by Western blot, and the expression levels of phosphorylated (P-) NF-κB P65 and phosphorylated (P-) STAT3 were detected.The concentrations of TNF-α, IL-12, IL-6 and IFN-γ in supernatant of hepatocellular carcinoma cell culture were determined by ELISA.On the basis of siNOD2 treatment, hepatocellular carcinoma cells were treated with NF-κB/STAT3 activator recombinant human lipocalin-2 protein (siNOD2+ RHlipocalin-2 group).The proliferation rate, apoptosis rate and inflammatory factor levels of hepatocellular carcinoma cells were measured.Results Compared with normal hepatocytes, NOD2 expression level in hepatocellular carcinoma cells was significantly up-regulated (P＜0.05).Compared with siNC group, NOD2, P-NF -κB P65 and P-STAT3 expression levels in siNOD2 group were significantly down-regulated (all P＜0.05), cell proliferation rate was decreased (P＜0.05), and cell apoptosis rate was increased (P＜0.05).The levels of TNF-α, IL-12, IL-6 and IFN-γ in supernatant of cell culture were down-regulated (all P＜0.05).Compared with siNOD2 group, the expression levels of P-NF -κB P65 and P-STAT3 in siNOD2+ RHlipocalin-2 group were significantly up-regulated (all P＜0.05), the cell proliferation rate was increased (P＜0.05), and the cell apoptosis rate was decreased (P＜0.05).The levels of TNF-α, IL-12, IL-6 and IFN-γ in supernatant of cell culture were up-regulated (all P＜0.05).Conclusion NOD2 knockdown inhibits the inflammatory response of hepatocellular carcinoma cells by regulating the NF-κB/STAT3 pathway, providing a new potential target for HCC treatment