The anti-cancer activities of amide-linked local anesthetics have been demonstrated in various types of bulky/differentiated cancer cells. However, whether these anesthetics also affect biological functions of cancer stem cells is largely unknown. In this study, we systematically investigated the effects of three commonly used amide-linked local anesthetics (ropivacaine, lidocaine and bupivacaine) on leukemia stem cell (LSC) derived from two different leukemia diseases (acute myeloid leukemia, n = 8 and chronic myeloid leukemia, n = 8) as well as normal hematopoietic stem cell (HSC) derived from cord blood donors (n = 8) as comparison. We show that all three local anesthetics at clinically achievable concentrations significantly inhibit colony formation and serial replating of LSC in a dose-dependent manner, suggesting their inhibitory effects on LSC differentiation, proliferation and self-renewal. In addition, lidocaine and bupivacaine are more potent than ropivacaine. However, local anesthetics at the same concentrations do not affect LSC and HSC survival, demonstrating the differentiation and self-renewal as the primary effects of local anesthetics on LSC and HSC. Interestingly, local anesthetics display certain selectivity between LSC and HSC by having higher efficacy on LSC than HSC. Mechanism studies using both pharmacological and genetic approaches demonstrate that these local anesthetics target LSC via inhibiting Wnt/beta-catenin but not Hedgehog or NF-kappa B signaling. Our work is the first to demonstrate the possible influence of amide-linked local anesthetics on cancer as well as normal stem cells via inhibiting Wnt/beta-catenin signaling. Our findings contribute to the comprehensive understanding of potential implication of amide-linked local aesthesis in tumor biology. (C) 2018 Elsevier Inc. All rights reserved.