The camptothecin analogue topotecan (TPT) induces tumor cell apoptosis due to interference with topoisomerase I and is clinically used as a second-line chemotherapeutic in the treatment for metastasizing ovarian and small cell lung carcinoma. Based on the more recent finding of TPT-mediated inhibition of the transcription factor hypoxia-induced factor-1α, a hallmark of solid tumors, TPT, is currently tested in clinical trials for its suitability as a first-line chemotherapeutic for the treatment for various types of tumors. Due to the gained clinical interest in TPT and in light of its modulatory effect on signaling pathways, which are also of importance for immune cell functions, we asked for potential effects of TPT on dendritic cells (DCs), the main antigen-presenting cell population of the immune system. Here, we show that TPT at a therapeutically relevant dose partially activated monocyte-derived DCs as reflected by enhanced migratory activity, elevated expression of HLA-DR and costimulatory/maturation markers, and accordingly an increased allogenic CD4⁺ T cell stimulation. In marked contrast, TPT prevented full maturation of DCs stimulated with a cocktail of proinflammatory mediators, accompanied by somewhat lower upregulation of NF-κB factors p65 and RelB.