Fragile histidine triad (FHIT) is a tumor suppressor gene whose allelic loss is associated to a number of human cancers. FHIT protein acts as a diadenosine oligophosphate hydrolase, but its tumor suppressive activity appears as independent from its enzymatic activity. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) can induce apoptosis in the FHIT-negative non-small lung cancer cell line Calu-1. We generated four FHIT-inducible Calu-1 cell clones and demonstrated that FHIT expression was able to protect cells from TRAIL-induced apoptosis, without affecting TRAIL-receptors surface expression. FHIT-specific small interference RNA transfection of SV40-immortalized normal bronchial BEAS cells that show levels of FHIT protein comparable to those of normal bronchial cells, resulted in a significant increase of TRAIL-induced apoptosis. Of note, suramin-mediated inhibition of FHIT enzymatic activity also enhanced TRAIL-induced apoptosis. We conclude that FHIT expression in lung cancer cells is protective from TRAIL-induced apoptosis. Our data suggest that FHIT exerts this protective effect downstream TRAIL-receptors and likely requires its dinucleoside-triphosphate hydrolase activity. As TRAIL represents in the near future a good candidate for death ligands-based anticancer therapy, its potential therapeutic use should be envisaged as preliminary to molecular genetics interventions or drug-induced epigenetic modulations aimed to restoring FHIT gene expression levels in non-small cells lung tumors.