<p>Sepsis-induced acute lung injury (ALI) is a life-threatening condition associated with high mortality, yet the molecular mechanisms driving alveolar damage remain incompletely understood. Long non-coding RNA (lncRNA) TUG1 has been implicated in organ injury, but its specific role and regulatory network in septic ALI have not been fully elucidated.Using lipopolysaccharide (LPS)-treated mouse lung epithelial (MLE-12) cells and a murine model of ALI, we investigated the functional role of TUG1 through overexpression strategies. Gene expression, protein levels, inflammatory cytokines, and miRNA interactions were assessed via qPCR, Western blotting, ELISA, and dual-luciferase reporter assays. Bioinformatic analysis of public datasets (GSE241238, GSE48080) was performed to validate clinical relevance.TUG1 expression was significantly downregulated in LPS-induced ALI models. TUG1 overexpression mitigated inflammation and oxidative stress by acting as a competing endogenous RNA (ceRNA) for miR-222-3p, thereby derepressing CALM1. Activation of CALM1 subsequently engaged the PRKG1/RYR3 signaling cascade, leading to restoration of AQP5-mediated alveolar fluid clearance. Analysis of public datasets confirmed suppression of the TUG1/CALM1 axis in septic patients and revealed its association with adverse survival outcomes. While the preliminary sample sizes (<i>n</i> = 3 in vitro, <i>n</i> = 5 in vivo) limit the statistical power (post-hoc 0.65), the robust trends support the TUG1 axis as a potential target, though larger cohort validation is needed.This study identifies TUG1 as a potential modulator of sepsis-induced ALI through the miR-222-3p/CALM1/PRKG1/RYR3/AQP5 axis. These findings underscore the therapeutic potential of targeting TUG1 to alleviate septic lung injury. These findings identify TUG1 as a protective lncRNA that acts via the miR-222-3p/CALM1 axis to regulate calcium signaling and alveolar fluid clearance. Targeting this pathway may offer a novel therapeutic strategy for sepsis-induced ALI.</p>