Background: Tumour-initiating cells (TICs) account for chemoresistance, tumour recurrence and metastasis, and therefore represent a major problem in tumour therapy. However, strategies to address TICs are limited. Recent studies indicate Cdk5 as a promising target for anti-cancer therapy and Cdk5 has recently been associated with epithelial-mesenchymal transition (EMT). However, a role of Cdk5 in TICs has not been described yet. Methods: Expression of Cdk5 in human cancer tissue was analysed by staining of a human tissue microarray (TMA). Functional effects of Cdk5 overexpression, genetic knockdown by siRNA and shRNA, and pharmacologic inhibition by the small molecule roscovitine were tested in migration, invasion, cell death, and tumorsphere assays and in tumour establishment in vivo. For mechanistic studies, molecular biology methods were applied. Results: In fact, here we pin down a novel function of Cdk5 in TICs: knockdown and pharmacological inhibition of Cdk5 impaired tumorsphere formation and reduced tumour establishment in vivo. Conversely, Cdk5 overexpression promoted tumorsphere formation which was in line with increased expression of Cdk5 in human breast cancer tissues as shown by staining of a human TMA. In order to understand how Cdk5 inhibition affects tumorsphere formation, we identify a role of Cdk5 in detachment-induced cell death: Cdk5 inhibition induced apoptosis in tumorspheres by stabilizing the transcription factor Foxo1 which results in increased levels of the pro-apoptotic protein Bim. Conclusions: In summary, our study elucidates a Cdk5-Foxo1-Bim pathway in cell death in tumorspheres and suggests Cdk5 as a potential target to address TICs.