Given that colorectal cancer stem cells (CCSCs) play key roles in the tumor dormancy, metastasis, and relapse, targeting CCSCs is a promising strategy in cancer therapy. Here, we aimed to identify the new regulators of CCSCs and found that Cullin 4B (CUL4B), which possesses oncogenic properties in multiple solid tumors, drives the development and metastasis of colon cancer by sustaining cancer stem-like features. Elevated expression of CUL4B was confirmed in colon tumors and was associated with poor overall survival. Inhibition of CUL4B in cancer cell lines and patient-derived tumor organoids led to reduced sphere formation, proliferation and metastasis capacity. Mechanistically, CUL4B coordinates with PRC2 complex to repress miR34a expression, thus upregulates oncogenes including MYCN and NOTCH1, which are targeted by miR34a. Furthermore, we found that elevated CUL4B expression is associated with miR34a downregulation and upregulation of miR34a target genes in colon cancer specimens. Collectively, our findings demonstrate that CUL4B functions to repress miR34a in maintaining cancer stemness in CRC and provides a potential therapeutic target.