Abstract: Cancer stem cells(CSCs) are considered as a core cellular subpopulation that drives tumor initiation, metastasis, and recurrence. Their survival and stemness maintenance rely on a specific microenvironment, known as the niche. This niche is constructed by various cellular components and non-cellular matrices, and collectively maintains CSC self-renewal and stemness characteristics through multiple mechanisms, including the regulation of classical signaling pathways, extracellular matrix(ECM)-mediated mechanotransduction, and metabolic-epigenetic interplay. Furthermore, the niche forms a therapeutic resistance system that integrates both physical and chemical barriers. Despite encouraging progress in disrupting niche structure, blocking key signaling networks, alleviating the immunosuppressive environment, and inducing differentiation, major bottlenecks for clinical translation remain, including the high heterogeneity of CSCs, limited target availability, impeded drug delivery, and potential toxicity risks. With the integrated application of multi-omics and spatial technologies, and the rapid advances in artificial intelligence(AI)-assisted target and drug discovery as well as smart nanodelivery systems, precise targeting of the CSC niche is anticipated to achieve breakthroughs. This review systematically elucidates the key mechanisms of the CSC niche in tumor progression and therapeutic resistance, summarizes the latest therapeutic strategies and translational prospects of targeting the niche, aiming to provide a theoretical foundation and insights for developing novel anti-tumor therapeutics.