Disruption of the intestinal epithelial barrier (IEB) predisposes preterm infants to necrotizing enterocolitis (NEC). Recent studies highlight the critical role of the mechanosensitive ion channel Piezo1 in maintaining intestinal epithelial homeostasis. Here, we aimed to investigate the specific role and underlying mechanisms of Piezo1 in NEC. We found that Piezo1 expression was up-regulated in NEC patients and associated with IEB dysfunction. In experimental NEC mice, pharmacological activation of Piezo1 exacerbated IEB disruption and NEC progression, whereas intestinal epithelial cell (IEC)-specific Piezo1 knockout protected IEB integrity. Mechanistically, Piezo1 activation contributed to IEB disruption by inducing endoplasmic reticulum (ER) stress and apoptosis, while inhibition of ER stress alleviated these effects. Moreover, Piezo1 modulation affected the integrity of Caco-2 monolayer barrier, and Piezo1 knockdown or inhibition attenuated LPS-induced ER stress activation and barrier disruption. Inhibition of ER stress also alleviated Piezo1-induced apoptosis and cell-cell junction injury. Finally, chelation of cytoplasmic Ca2+ effectively attenuated Piezo1-induced ER stress and barrier disruption. Our study demonstrates that Piezo1-mediated Ca2+ influx triggers ER stress-dependent apoptosis, leading to IEB dysfunction in NEC. These findings suggest that targeting Piezo1 to protect IEB integrity may represent a promising therapeutic strategy for NEC.