Although several epigenetic modulating drugs are suggested to target cancer stem cells (CSCs), additional identification of anti-CSC drugs is still necessary. Here we showed that JIB-04, a pan-selective inhibitor of histone demethylase(s), was identified as a small molecule that selectively target colorectal CSCs. Our data showed that JIB-04 is capable of reducing self-renewal and stemness of colorectal CSCs in three different colorectal cancer cell lines. JIB-04 significantly attenuated CSC tumorsphere formation, growth/relapse, invasion, and migration in vitro. Furthermore, JIB-04-treated colorectal cancer cells showed reduced tumorigenic activity in vivo. RNA sequencing analysis revealed that JIB-04 affected various cancer-related signaling pathways, especially Wnt/β-catenin signaling, which is crucial for the proliferation and maintenance of colorectal cancer cells. qRT-PCR and TOP/FOP flash luciferase assays showed that JIB-04 down-regulated the expression of Wnt/β-catenin-regulated target genes associated with colorectal CSC function. Overall, the effects of JIB-04 were equal to or greater than those of salinomycin, a known anti-colorectal CSC drug, despite the lower concentration of JIB-04 compared with that of salinomycin. Our results strongly suggest that JIB-04 is a promising drug candidate for colorectal cancer therapy.