Various Long non-coding RNAs (lncRNAs) and MicroRNAs (miRNAs) have been demonstrated to be involved in colorectal cancer stem cells (CSCs). WBSCR22 is a key gene we previously found that functions in colorectal cancer (CRC). This paper aims to investigate the effects of WBSCR22 and its corresponding miRNA and lncRNA in CRC. The expression of WBSCR22 was detected by Western blot and qRT-PCR analysis. Tumor sphere formation assays, CCK-8 analysis, and Transwell assays were applied to examine the colorectal cancer stem cell properties. Luciferase assay and biotin RNA pulldown assay were used to detect the interaction between miR-509-5p and WBSCR22/Linc00346. WBSCR22 is highly expressed in CRC and correlates with poor prognosis. Knockdown of WBSCR22 decreased the sphere-forming capacities, cell proliferation, and invasion abilities. WBSCR22 was negatively regulated by miR-509-5p. Linc00346 promoted the expression of WBSCR22 by adsorbing miR-509-5p. The Linc00346/miR-509-5p/WBSCR22 signal axis regulated the characteristics of colon cancer stem cells. Linc00346 regulated the expression of WBSCR22 by binding to miR-509-5p, thereby monitoring the characteristics of colorectal cancer stem cells. Thus, WBSCR22, Linc00346, and miR-509-5p might be utilized as potential targets for clinical diagnosis and treatment of CRC.