PURPOSE: TNF-related apoptosis inducing ligand (TRAIL) expression by immune cells contributes to anti-tumor immunity. A naturally occurring splice variant of TRAIL, called TRAILshort, antagonizes TRAIL-dependent cell killing. It is unknown whether tumor cells express TRAILshort and if it impacts anti-tumor immunity. EXPERIMENTAL DESIGN: We used an unbiased informatics approach to identify TRAILshort expression in primary human cancers, and validated those results with immunohistochemistry (IHC) and in situ hybridization (ISH). TRAILshort specific monoclonal antibodies were used to determine the effect of TRAILshort on tumor cell sensitivity to TRAIL, and to immune effector cell dependent killing of autologous primary tumors. RESULTS: As many as 40% of primary human tumors express TRAILshort by both RNAseq and IHC analysis. By ISH, TRAILshort expression is present in tumor cells and not bystander cells. TRAILshort inhibition enhances cancer cell lines sensitivity to TRAIL dependent killing both in vitro and in immunodeficient xenograft mouse models. Immune effector cells isolated from patients with B cell malignancies killed more autologous tumor cells in the presence compared to the absence of TRAILshort antibody (P<0.05). CONCLUSION: These results identify TRAILshort in primary human malignancies, and suggest that TRAILshort blockade can augment the effector function of autologous immune effector cells.