Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse reactions in chemotherapy with cytotoxic anticancer agents. We have demonstrated that CIPN caused by paclitaxel involves high mobility group box 1 (HMGB1), a nuclear protein, derived from macrophages (Mφ) in laboratory animals (Neuropharmacology 141, 201, 2018), and that postmenopausal estrogen decline aggravates CIPN in patients undergoing paclitaxel-based chemotherapy (J Pharmacol Sci 146, 49, 2021). In this study, we analyzed estrogen modulation of upstream and downstream mechanisms of HMGB1. Female mice were subjected to ovariectomy or sham operation, and received repeated paclitaxel treatment. Nociceptive threshold in the hindpaw was assessed by the von Frey test. HMGB1 release from Mφ-like RAW264.7 cells was determined by Western blotting. Repeated treatment with paclitaxel at 1 mg/kg, a subeffective dose, caused CIPN in ovariectomized, but not sham-operated, female mice, an effect prevented by repeated 17β-estradiol (E2) treatment. Paclitaxel produced cytoplasmic translocation and extracellular release of nuclear HMGB1 in Mφ-like RAW264.7 cells, which were suppressed by E2. The effects of E2 in RAW264.7 cells were reversed by each of the antagonists of ERα and GPR30, nuclear and membrane estrogen receptors, respectively. Intraplantar (i.pl.) administration of HMGB1 at 10-100 ng decreased nociceptive threshold in naïve female mice. HMGB1, administered i.pl. at 1 ng, caused mechanical allodynia in ovariectomized, but not sham-operated, mice, an effect reversed by repeated systemic preadministration of E2. Our data suggest that estrogen suppresses both paclitaxel-induced HMGB1 release from Mφ, and the HMGB1-induced pain signaling, thereby playing a protective role against CIPN development following paclitaxel treatment.