B-cell receptor-associated protein 31 (BAP31) is an endoplasmic reticulum (ER) membrane protein which plays a role as a molecular chaperone for the newly synthesized transmembrane proteins. BAP31 is also an important apoptosis regulator for extrinsic apoptosis induction in the ER membrane. Recent studies have shown that BAP31 is also expressed on the surface of embryonic stem cells. However, the function of cell surface BAP31 (csBAP31) still remains unclarified. In an attempt to search for surface markers on tumorspheres, here, we generated monoclonal antibodies (MAbs) against the sphere cells from the non-small cell lung carcinoma cell (NSCLC) line A549. SP1-B7, one of the MAbs, recognized csBAP31 whose expression was further increased on A549 sphere cells, as compared with A549 adherent cells. To investigate the role of csBAP31 in A549 cells, A549 adherent and sphere cells were stained with annexin V, propidium iodide, and SP1-B7. Interestingly, annexin V-high cells showed increased expression of csBAP31 as compared with annexin V-low cells. Caspase-3/7 activity was also increased in csBAP31-high cells as compared with csBAP31-low cells, suggesting that csBAP31-high cells are more sensitive to apoptosis. To further demonstrate the survival of csBAP31-positive A549 cells, csBAP31-positive and -negative A549 cells were sorted and subjected to the clonogenic survival assay. The colony number of csBAP31-positive A549 cells was decreased by approximately 1.7-fold, as compared that of csBAP31-negative A549 cells, suggesting that csBAP31-positve cells are sensitive to cell death indeed. The results suggest that enhanced expression of csBAP31 contributes to poor survival of NSCLC cells.