Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here, we show for the first time that G2.2, a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide, selectively inhibits colonic CSCs in vivo G2.2-reduced CSCs (CD133+/CXCR4+, Dual hi) induced HT-29 and HCT 116 colon xenografts' growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact, G2.2 robustly inhibited CSCs' abundance (measured by levels of CSC markers, e.g., CD133, DCMLK1, LGR5, and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly, G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly, G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions, blood coagulation profile, and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro, in vivo, and ex vivo mechanistic studies, we showed that G2.2's inhibition of CSC self-renewal was mediated through activation of p38$\alpha$, uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence, G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs.