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The membrane protein CD24 represents a promising immunotherapeutical approach. The present study aimed to decipher the molecular function of CD24 <jats:italic>in vitro<\/jats:italic> and evaluate the cytotoxic capacity of a third\u2010generation natural killer (NK) cell chimeric antigen receptor (CAR) against CD24 in urologic tumour cell lines. Up to 20 urologic tumour cell lines and several non\u2010malignant control cells were included. XTT viability assays and annexin V\/propidium iodide flow cytometry analyses were performed to measure cell viability and apoptosis rates, respectively. Co\u2010immunoprecipitation followed by mass spectrometry analyses identified direct interaction partners of CD24. Luciferase reporter assays were used to functionally validate transactivation of <jats:italic>CD24<\/jats:italic> expression by SOX2. <jats:italic>N<\/jats:italic>\u2010\u00a0and <jats:italic>O<\/jats:italic>\u2010glycosylation of CD24 were evaluated by enzymatic digestion and mass spectrometry. The study demonstrates that SOX2 transactivates <jats:italic>CD24<\/jats:italic> expression in embryonal carcinoma cells. In cells of different urological origins, CD24 interacted with proteins involved in cell adhesion, ATP binding, phosphoprotein binding and post\u2010translational modifications, such as histone acetylation and ubiquitination. Treatment of urological tumour cells with NK\u2010CD24\u2010CAR cells resulted in a decreased cell viability and apoptosis induction specifically in CD24<jats:sup>+<\/jats:sup> tumour cells. Limitations of the study include the <jats:italic>in vitro<\/jats:italic> setting, which still has to be confirmed <jats:italic>in vivo<\/jats:italic>. 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