Cancer stem cells (CSCs), or cancer cells with stem cell properties, are a rare population of tumor bulk and are recognized to be responsible for cancer recurrence, drug resistance, and metastasis. However, the molecular mechanisms of how to regulate the differentiation and self-renewing of CSCs are poorly understood. Krüppel-like factors (KLFs) are essential DNA-binding transcriptional regulators with diverse functions in various cellular processes, including differentiation, proliferation, inflammation, migration, and pluripotency. Recent progress has highlighted the significance of KLFs in tumor progression and CSCs. The regulatory functions of KLFs in the development of cancer and CSCs have become a burgeoning area of intense research. In this review, we summarize the current understanding and progress of the transcriptional regulation of KLFs in CSCs and discuss the functional implications of targeting CSCs by KLFs for cancer therapeutics.