Ferroptosis plays a critical role in LPS-induced acute lung injury and is modulated by endoplasmic reticulum stress (ERS). As a typical ER stress-responsive protein, recently mesencephalic astrocyte-derived neurotrophic factor (MANF) has been demonstrated to attenuate LPS-induced acute lung injury (ALI) through repressing macrophage activation. However, whether MANF exerts a preventive role on ferroptosis and excess ER stress remains unclear. Here, we first built a protein-protein interaction (PPI) network to obtain potential interacting proteins related to MANF through STRING and GeneMANIA. Then, male C57BL/6J mice were used to build a model of LPS-induced lung injury. Two days before LPS injection, the tail vein injected recombinant murine MANF (rmMANF) at 750 μg/kg. Twenty-four hours after the LPS injection, the histopathological changes and damage in the lung tissues were detected and scored by HE staining and TUNEL assay, respectively. Endogenous MANF levels, oxidative stress markers (GSH, SOD, CAT, and MDA), ERS markers (GRP78, PERK, and ATF4), and the ferroptosis markers (iron, GPX4, and 4-HNE) in the lung tissues were measured by IHC, western blotting, and commercial kits. Our results showed that LPS induced significant lung injury to the increase in MPO, MDA, and 4-HNE, a decrease in GPX4 and GSH, SOD, CAT, and total iron accumulation in LPS-exposed mice. Simultaneously, GRP78/PERK/ATF4 pathway was notably activated by LPS, accompanied by the down-regulation of MANF. Furthermore, rmMANF pretreatment markedly prevented LPS-induced lung tissue injury and ferroptosis characteristics with the increased GPX4 level in sepsis mice. Finally, we found that LPS-induced oxidative stress and activation of the GRP78/PERK/ATF4 pathway were significantly restrained by rmMANF pretreatment, except for endogenous MANF level. Overall, rmMANF pretreatment can prevent sepsis-associated lung injury by inhibiting ER stress-induced ferroptosis in mice.