In this study, we report enrichment of the lncRNA RBM5-AS1/LUST during sphere formation of colon CIC. Its silencing impaired WNT signaling, whereas its overexpression enforced WNT signaling, cell growth, and survival in serum-free media. Mechanistic investigations showed that silencing or overexpression of RBM5-AS1 caused a respective loss or retention of B-catenin from TCF4 complexes bound to the WNT target genes SGK1, YAP1, and MYC Our work suggests that RBM5-AS1 activity is critical for the functional enablement of colon cancer stem-like cells. Furthermore, it defines the mechanism of action of RBM5-AS1 in the WNT pathway via physical interactions with B-catenin, helping organize transcriptional complexes that sustain colon CIC function.