Ferroptosis, an iron-dependent type of cell death, has attracted significant attention in recent years. Accumulating evidence demonstrates that ferroptosis critically contributes to the pathogenesis of intestinal ischemia/reperfusion (I/R) primarily through triggering excessive lipid peroxidation, disrupting iron homeostasis, and disabling endogenous antioxidant system. Therefore, targeted inhibition of ferroptosis represents a promising therapeutic strategy to alleviate intestinal I/R damage. However, the upstream molecular regulatory networks governing ferroptosis in intestinal I/R, especially miRNA-mediated post-transcriptional regulatory mechanisms, are not fully understood. MiRNA microarray analysis revealed that the expression of miR-381-3p is upregulated following intestinal I/R insult. Bioinformatic prediction combined with dual-luciferase reporter assays confirmed that miR-381-3p directly targets and negatively regulates cAMP response element-binding protein 1 (CREB1), a key transcription factor closely implicated in ferroptosis modulation. Subsequent experiments confirmed that CREB1 specifically binds to the promoter region of peroxiredoxin 6 (PRDX6) to facilitate its transcriptional activation, as quantified by quantitative real-time PCR. Both in vivo intestinal I/R injury mouse models and an in vitro Caco-2 cell hypoxia/reoxygenation (H/R) model were utilized for functional verification. The results revealed that upregulation of the CREB1/PRDX6 axis significantly suppressed the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), which is a key marker of ferroptosis. Meanwhile, enhanced GPX4 activity and elevated intracellular glutathione (GSH) content were observed, accompanied by reduced intracellular iron overload and alleviated histological and cellular morphological damage. Conversely, inhibition of the CREB1/PRDX6 axis exacerbated ferroptosis, with corresponding adverse changes in these ferroptosis-related indicators. In conclusion, the miR-381-3p/CREB1/PRDX6 signaling axis protects against intestinal I/R by suppressing ferroptosis, providing novel molecular targets and a theoretical basis for the prevention of intestinal I/R injury.