Ca²⁺-activated K⁺ channel K_Ca1.1 has a potential as a prognostic tumor marker in several solid cancers. Three-dimensional (3D) in vitro cell culture system mimics in vivo solid tumors resistance to chemotherapy in tumor microenvironment (TME). In the MG-63 cells isolated from 3D spheroid models, K_Ca1.1 activator-induced hyperpolarizing responses were largely enhanced, compared with adherent 2D monolayer cells. In lipid-raft-enriched compartments of MG-63 spheroids, the protein expression level of K_Ca1.1 was significantly increased, without changing its transcriptional level. The spheroid formation caused down-regulation of the ubiquitin E3 ligase FBXW7, and its inhibition in 2D monolayer cells increased the K_Ca1.1 protein expression. In the MG-63 spheroids, treatment with the K_Ca1.1 inhibitor suppressed chemoresistance ability to paclitaxel, doxorubicin, and cisplatin. Of several multidrug resistance ABC transporters, a multidrug resistance-associated protein, MRP1 was up-regulated in the MG-63 spheroids, and chemosensitivity was recovered by the K_Ca1.1 inhibition. Taken together, pharmacological inhibition of K_Ca1.1 may be an attractive new strategy for conquest of chemotherapeutics synergizing in the hypoxic TME of K_Ca1.1-positive solid cancers.