Abstract: Melatonin (5–15 mg/kg) protects male animals against ischemic stroke. We explored the potential interactions and synergistic neuroprotection of melatonin and estrogen using a panel of lipid peroxidation and radical-scavenging assays, primary neuronal cultures subjected to oxygen–glucose deprivation (OGD), and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Neuroprotective efficacy of melatonin was also evaluated in both reproductively active and ovariectomized female rats subjected to transient focal cerebral ischemia. Relative to melatonin or estradiol (E2) alone, a combination of the two agents exhibited robust, synergistic antioxidant and radical-scavenging actions (P < 0.05, respectively). Additionally, the two agents, when combined at large doses, showed synergistic inhibition in the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in the LPS-stimulated RAW 264.7 cells (P < 0.05, respectively). Alternatively, co-treatment with melatonin and E2 independently, but not combined, showed a U-shaped dose-responsive (hormetic) cytoprotection for neuronal cultures subjected to OGD. When combined at a dosage either positively or negatively skewed from each optimal dosage, however, co-treatment caused synergistic neuroprotection. Relative to vehicle-injected controls, melatonin given intravenously at 1–5 mg/kg, but not 0.1 or 15 mg/kg, significantly reduced brain infarction and improved neurobehavioral outcomes (P < 0.05, respectively) in reproductively active female rats. In ovariectomized stroke rats, melatonin was only effective at a large dosage (15–50 mg/kg). These results demonstrate complex interactions and synergistic antioxidant, radical-scavenging, and anti-inflammatory actions between estradiol and melatonin, and highlight the potential need to rectify the melatonin’s hormetic dose–response by the level of circulating estradiol in the treatment of female stroke patients.