Heat stroke (HS) is a severe condition associated with prolonged exposure to high temperatures, leading to systemic inflammation and multi-organ damage. Disruption of the intestinal barrier plays a crucial role in HS progression by allowing endotoxins to enter circulation and trigger widespread inflammation. Lactoferrin (LF), known for its immune-modulating and barrier-protective properties, shows promise in mitigating HS-related damage. This study investigated the protective effects of LF on the intestinal barrier under HS conditions using in vitro and in vivo models. In Caco-2 cell monolayers exposed to heat stress, LF improved barrier integrity by increasing transepithelial electrical resistance (TEER) and maintaining tight junctions. In mice, LF supplementation enhanced heat tolerance, delayed HS onset, and reduced multi-organ damage, whereas LF knockout (KO) mice exhibited exacerbated damage. Further analysis revealed that heat stress induced ferroptosis by suppressing GPX4 and SLC7A11, contributing to intestinal injury. LF alleviated ferroptosis by upregulating these proteins and reducing Fe²⁺ accumulation, reactive oxygen species (ROS) generation, and lipid peroxidation. Additionally, LF inhibited heat stress-induced activation of the MAPK pathway by suppressing the phosphorylation of p38, ERK, and JNK, suggesting that MAPK signaling mediates its protective effects. These findings suggest that LF supplementation may enhance heat tolerance and prevent HS-induced intestinal barrier damage through ferroptosis inhibition and MAPK pathway regulation.