Aim: As our understanding of cancer stem cell (CSC) biology improves, search for inhibitory agents of CSCs and metastatic CSCs (mCSCs) positive for CXCR4 is warranted. Withaferin A (WA), a withanolide extracted from the medicinal plant Withania somnifera, has been shown to exhibit anti-cancer effects through multiple mechanisms. Whether WA could selectively target CSCs, mCSCs, or non-CSCs of a gastrointestinal (GI) carcinoma tumor remains unclear.Methods: Side-population (SP) analysis, ﬂow cytometric phenotyping and sorting, non-invasive imaging in conjunction with xenotransplantation, and immunohistology were used in this investigation.Results: Using the lymph node metastatic GI cancer cell line UP-LN1, consisting of CD44high/CD24low ﬂoating (F) and CD44low/CD24high adherent (A) cell subsets, this study demonstrated that as compared with parental UP-LN1 cells or A cells, WA preferentially reduced F-cell proliferation, tumor sphere formation, and SP cells in vitro in greater efﬁ ciencies by apoptosis. This action was mechanistically mediated via the down-regulation of CXCR4/CXCL12 and STAT3/interleukin-6 axes, both of which are instrumental in the acquisition of metastatic ability. Attenuation of interferon-γ-induced CXCR4 expression in F cells by knockdown with siRNA or blocking with an anti-CXCR4 antibody, followed by Western blot analysis, showed signiﬁ cantly reduced metastatic potential in vitro. The extent of in vitro anti-invasive effect of WA on the IFN-γ-treated F cells was signiﬁ cantly greater than on the F cells without WA treatment, or F cells treated with control siRNA or with control IgG antibody. The observed in vitro effects of WA on the CSC and mCSC targeting were validated by data obtained with non-invasive imaging in NOD/SCID mouse xenotransplantation.Conclusion: WA could efﬁ ciently block the formation of both CSCs and mCSCs in the UP-LN1 cell line, suggesting that WA may be considered an effective therapeutic agent for this type of GI malignancies.