The CD56 antigen (NCAM-1) is highly expressed on several malignancies with neuronal or neuroendocrine differentiation, including small-cell lung cancer and neuroblastoma, tumor types for which new therapeutic options are needed. We hypothesized that CD56-specific chimeric antigen receptor (CAR) T cells could target and eliminate CD56-positive malignancies. Sleeping Beauty transposon-generated CD56R-CAR T cells exhibited αβT-cell receptors, released antitumor cytokines upon co-culture with CD56⁺ tumor targets, demonstrated a lack of fratricide, and expression of cytolytic function in the presence of CD56⁺ stimulation. The CD56R-CAR⁺ T cells are capable of killing CD56⁺ neuroblastoma, glioma, and SCLC tumor cells in in vitro co-cultures and when tested against CD56⁺ human xenograft neuroblastoma models and SCLC models, CD56R-CAR⁺ T cells were able to inhibit tumor growth in vivo. These results indicate that CD56-CARs merit further investigation as a potential treatment for CD56⁺ malignancies.