Objective Investigating the effects of matrix stiffening on hepatocyte inflammation and the related molecular mechanisms. Methods Alginate/gelatin hydrogel spheres were constructed by physical cross-linking method and the stiffness was measured by compression test. Human normal liver cells HL7702 were cultured in alginate/gelatin hydrogel spheres with stiffness conditions of 5, 15, and 30 kPa for 48 h. The express levels of inflammatory factor IL-6 in hepatocytes were investigated by Western Blot assay. The extent of hepatocyte DNA damage was detected by comet assay and the express levels of DNA double-strand break marker proteins γ-H2AX and 53BP1 were explored by Western Blot and Immunofluorescence assays. The role of cGAS-STING signaling pathway in the regulation of matrix stiffening on the expression of inflammatory factors in hepatocytes were explored by Western Blot assay. Results (1) Matrix stiffening promotes the expression levels of IL-6 in hepatocytes. (2) Matrix stiffening induces hepatocyte DNA damage while the expression levels of γ-H2AX and 53BP1 were significantly up-regulated. (3) Matrix stiffening activates cGAS-STING signaling pathway in hepatocytes. (4) Inhibition of STING significantly reduces the expression of IL-6 in hepatocytes cultured under stiffened matrices. Conclusion Matrix stiffening induces DNA damage to activate the cGAS-STING signaling pathway to promote inflammatory factors expression in hepatocytes. This study provides a mechanobiological rationale for targeting matrix stiffness to treat HCC and offers key experimental support for new clinical strategies against HCC. These results provide an experimental basis for a deeper understanding of the mechanobiological mechanisms in liver lesions, and also provide experimental evidence for liver cell injury therapy targeting matrix stiffness.