Background <p>Iron overload is a pathological condition characterized by excessive iron accumulation, leading to oxidative stress and tissue damage. Due to the absence of active iron excretion pathways in mammals, regulating intestinal iron absorption remains a key therapeutic strategy. α-Lipoic acid (ALA), a potent antioxidant, has shown protective effects against iron overload toxicity, but its role in modulating duodenal iron absorption has not been fully elucidated. This study investigated the effects and mechanisms of ALA on duodenal iron absorption in a rat model of iron overload.</p> Methods and results <p>Thirty-two 7-week-old male Sprague Dawley rats were randomly divided into four groups: control (CON), ALA, iron overload (IO), and ALA and iron overload (ALA + IO). The results showed that ALA administration significantly improved growth performance and attenuated iron accumulation in plasma, liver, and spleen. Histological analysis revealed that ALA alleviated iron-induced shortening of duodenal villi and suppressed iron deposition in the duodenum. Mechanistically, ALA Markedly reduced the protein levels of divalent metal transporter 1 (DMT1), a critical iron transporter for duodenal non-heme iron absorption, without affecting its mRNA expression, suggesting post-transcriptional regulation. Bioinformatic and dual-luciferase assays identified miR-15b-5p and miR-195-5p as direct regulators of DMT1 presence of an iron-responsive element (IRE) (DMT1 + IRE). Notably, ALA restored the expression of these microRNAs (miRNAs), which were downregulated under iron overload conditions.</p> Conclusions <p>These findings indicate that ALA suppresses duodenal non-heme iron absorption via miRNA-mediated post-transcriptional repression of DMT1, offering a potential therapeutic strategy for iron overload-associated complications.</p>