BackgroundOvarian cancer, particularly epithelial ovarian cancer (EOC), is the leading cause of cancer-related mortality among women. Our previous study revealed that high HMGB3 levels are associated with poor prognosis and lymph node metastasis in patients with high-grade serous ovarian carcinoma; however, the role of HMGB3 in EOC proliferation and metastasis remains unknown.MethodsMTT, clonogenic, and EdU assays were used to assess cell proliferation. Transwell assays were performed to detect cell migration and invasion. Signaling pathways involved in HMGB3 function were identified by RNA sequencing (RNA-seq). MAPK/ERK signaling pathway protein levels were evaluated by western blot.ResultsHMGB3 knockdown inhibited ovarian cancer cell proliferation and metastasis, whereas HMGB3 overexpression facilitated these processes. RNA-seq showed that HMGB3 participates in regulating stem cell pluripotency and the MAPK signaling pathway. We further proved that HMGB3 promotes ovarian cancer stemness, proliferation, and metastasis through activating the MAPK/ERK signaling pathway. In addition, we demonstrated that HMGB3 promotes tumor growth in a xenograft model via MAPK/ERK signaling.ConclusionsHMGB3 promotes ovarian cancer malignant phenotypes and stemness through the MAPK/ERK signaling pathway. Targeting HMGB3 is a promising strategy for ovarian cancer treatment that may improve the prognosis of women with this disease.Video AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s12964-023-01172-7.