Lung adenocarcinoma (LUAD) is a leading cause of cancer-related mortality, underscoring the urgent need for novel biomarkers and therapeutic targets. Through transcriptomic analysis of 4456 genes in TCGA-LUAD cohorts, we identified RBMS3 as a significantly downregulated tumor suppressor (log2 fold change = -1.82, adjusted P = 3.2 × 10⁻5). Clinically, elevated RBMS3 expression was independently associated with improved overall survival (HR = 0.766, 95% CI 0.602-0.973, P = 0.029), validated by Kaplan-Meier analysis (Log-rank P = 0.027). Functionally, RBMS3 overexpression in A549 and PC-9 LUAD cells suppressed invasion (66.53% and 52.46% reduction, respectively; P < 0.01) and induced apoptosis (total apoptosis increased by 6.53% and 8.57%; P < 0.05). Cell cycle analysis revealed accelerated G1-to-S phase transition, with G1-phase proportions decreasing from 44.6 to 36.87% in A549 (P < 0.01) and from 49.83 to 37.13% in PC-9 (P < 0.01). TIMER-based correlation analysis demonstrated a positive association between RBMS3 expression and immune cell infiltration, with the regression line indicating significant correlations for B cells (cor = 0.16, P = 4.25 × 10⁻4), CD8 + T cells (cor = 0.214, P = 1.86 × 10⁻⁶), CD4 + T cells (cor = 0.24, P = 8.99 × 10⁻⁸), macrophages (cor = 0.341, P = 1.07 × 10⁻14), neutrophils (cor = 0.277, P = 5.71 × 10⁻10), and dendritic cells (cor = 0.369, P = 3.70 × 10⁻17). These findings underscore RBMS3's dual role in LUAD as a tumor suppressor and immune microenvironment modulator, offering novel insights for prognosis and therapy.© 2025. The Author(s).