Background: As a component of nucleosomes, histone H3 plays an important role in chromosome structure and gene expression. Current studies have mostly focused on the role of histones in epigenetics, but in addition to this, the role of histones themselves in tumor development and microenvironment have been less explored. Methods: Western blot and immunofluorescence were carried out to detect the content and localization of histone H3 in hepatocellular carcinoma. The changes of histone H3 were observed in hypoxia treatment cells, the specific action mechanism of histone H3 was studied by CoIP and other methods. Cell Counting Kit-8 assay, plate cloning assay and transwell assay were used to exam the effect of histone H3 on cell proliferation and metastasis, which were verified by subcutaneous tumors in mice and lung metastasis by tail vein injection in mice. Results: We found that histone H3 was overexpressed in hepatocellular carcinoma tumor tissues compared to adjacent non-tumor tissues, and there was concomitant translocation of histone H3 from the nucleus to the cytoplasm. We found that hypoxia could contribute to this phenomenon of histone H3 translocation from the nucleus to the cytoplasm in hepatocellular carcinoma cells and increased binding levels to TLR9. At the same time, hypoxia induced downstream activation of TLR9 and caspase-1, as well as cleavage and release of the pro-inflammatory cytokines IL-1β and IL-18. We further demonstrated that histone H3 could also promote proliferation and metastasis of hepatocellular carcinoma through TLR9 activation of NLRP3 inflammasome. In addition, overexpression of histone H3 was also confirmed to promote hepatocellular carcinoma proliferation and metastasis in mouse models of hepatocellular carcinoma growth assay and lung metastasis. Conclusions: In hypoxic hepatocellular carcinoma cells, histone H3 can translocate to the cytoplasm and activate caspase-1 via TLR9, thereby producing pro-inflammatory cytokines that promote tumor proliferation and metastasis.© The author(s).