Purpose Oridonin, a bioactive diterpenoid derived from Rabdosia rubescens , has been widely reported to exhibit anticancer activity in multiple types of cancer. However, the molecular mechanism of oridonin in human laryngeal carcinoma has not been clearly elucidated. This study investigated the function of oridonin in laryngeal carcinoma to provide a research basis for laryngeal carcinoma therapy. Methods The proliferation of laryngeal carcinoma Hep-2 and TU212 cells treated with oridonin was determined by MTT assay. The apoptotic induction effect of oridonin on Hep-2 and TU212 cells was analyzed by flow cytometry, Western blot analysis and caspase3 activity assay. In addition, the caspase inhibitor, Z-VAD-fmk, was synergistically treated with oridonin to detect the function of caspase cascade in oridonin-mediated apoptosis. Then, the expressions of endoplasmic reticulum (ER) stress-related proteins (GRP78, phosphorylated-PERK, phosphorylated-eIF2α and CHOP) were measured in Hep-2 and TU212 cells by Western blotting. The cells were treated with 4-PBA (an ER stress inhibitor) or knockdown of CHOP to explore the role of ER stress in oridonin-mediated apoptosis in laryngeal carcinoma. Subsequently, a nude mouse xenograft model was constructed to confirm the function of oridonin in laryngeal carcinoma in vivo. Results Oridonin was found to significantly inhibit the proliferation of laryngeal carcinoma Hep-2 and TU212 cells in a concentration-dependent manner. Then, we confirmed that oridonin could induce apoptosis in human laryngeal carcinoma cells. The caspase inhibitor, Z-VAD-fmk, could partially reverse the pro-apoptotic effect of oridonin on human laryngeal carcinoma cells. Subsequently, Western blotting analysis demonstrated that endoplasmic reticulum (ER) stress-related proteins (GRP78, phosphorylated-PERK, phosphorylated-eIF2α and CHOP) were up-regulated in Hep-2 and TU212 cells exposed to oridonin. In addition, 4-PBA (an ER stress inhibitor) or knockdown of CHOP could antagonize oridonin-induced apoptosis. Oridonin significantly decreased the tumorigenicity of Hep-2 cells in a nude mouse xenograft model. Conclusion Oridonin-induced apoptosis of human laryngeal carcinoma through the activation of ER stress.