Ischemic stroke is a common event of cerebrovascular disease, and inhibiting ferroptosis is associated with improvement in ischemic stroke. The purpose was to evaluate the effects of activating transcription factor 3 (ATF3) knockdown on ferroptosis in ischemic stroke and to reveal its underlying mechanism. Ischemic condition was simulated in HT22 cells by oxygen glucose deprivation/reperfusion (OGD/R), and an ischemic stroke model was induced by middle cerebral artery occlusion (MCAo) in rats. ATF3 was knocked down to investigate its effects on cerebral infarct size, cell death, oxidative stress, Fe and ferroptosis, and the kelch-like ECH-Associating protein 1 (Keap1)/nuclear factor erythroid 2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in HT22 cells and stroke rats. Knockdown of ATF3 decreased neurological score, increased duration on the rotarod, and decreased scores of balance beam test. Meanwhile, knockdown of ATF3 decreased infarct area, suppressed apoptosis, and improved brain structure in MCAo rats. Knockdown of ATF3 decreased the levels of malondialdehyde (MDA), reactive oxygen species (ROS) and Fe, increased glutathione (GSH), decreased cyclooxygenase 2 (COX2) and increased glutathione peroxidase 4 (GPX4). The levels of Nrf2 and HO-1 were upregulated while Keap1 was downregulated after knocking down ATF3. Similarly, knockdown of ATF3 alleviated ischemic condition, inhibited ferroptosis, reduced cell apoptosis, and activated the Keap1/Nrf2/HO-1 pathway in HT22 cells. ML385 reversed the effects of ATF3 knockdown on ferroptosis and Keap1/Nrf2/HO-1 pathway. Knockdown of ATF3 alleviates ischemic stroke and inhibits ferroptosis via activating the Keap1/Nrf2/HO-1 pathway, which provides theoretical supports for ischemic stroke.