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As the sentinels of innate and adaptive immune system, dendritic cells (DCs) have been considered to hold a great promise for medical application. Among the diverse types of DCs, monocyte-derived DCs (mo-DCs) generated in vitro have been most commonly employed. We have been improving the culture protocol and devised a protocol to produce mature interferon-α-induced DCs (IFN-DCs), hereinafter called (mat)IFN-DCs. While exploring the relationship between the expression of CD56 and the cytotoxic activity of (mat)IFN-DCs, we unexpectedly found that sorting of (mat)IFN-DCs with CD56 antibody-coated microbeads (MB) resulted in fractionating cells with tumoricidal activity into the flow-through (FT) but not MB-bound fraction. We uncovered that the FT fraction contains cells expressing low but substantial level of CD56. Moreover, those cells express granzyme B (GrB), perforin (PFN), and serpin B9 at high levels. By employing a specific inhibitor of PFN, we confirmed that direct tumoricidal activity relies on the GrB/PFN pathway. We designated subpopulation in FT fraction as CD56 dim and that in CD56 positively sorted fraction as CD56 bright , respectively. This is the first time, to our knowledge, to identify subpopulations of CD56-positive IFN-DCs with distinct tumoricidal activity which is ascribed to high expression of the components of GrB/PFN pathway., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)