The large-conductance Ca -activated K channel K 1.1 plays a pivotal role in tumor development and progression in several solid cancers. The three-dimensional (3D) in vitro cell culture system is a powerful tool for cancer spheroid formation, and mimics in vivo solid tumor resistance to chemotherapy in the tumor microenvironment (TME). K 1.1 is functionally expressed in osteosarcoma and chondrosarcoma cell lines. K 1.1 activator-induced hyperpolarizing responses were significantly larger in human osteosarcoma MG-63 cells isolated from 3D spheroid models compared with in those from adherent 2D monolayer cells. The present study investigated the mechanisms underlying the upregulation of K 1.1 and its role in chemoresistance using a 3D spheroid model. K 1.1 protein expression levels were significantly elevated in the lipid-raft-enriched compartments of MG-63 spheroids without changes in its transcriptional level. 3D spheroid formation downregulated the expression of the ubiquitin E3 ligase FBXW7, which is an essential contributor to K 1.1 protein degradation in breast cancer. The siRNA-mediated inhibition of FBXW7 in MG-63 cells from 2D monolayers upregulated K 1.1 protein expression. Furthermore, a treatment with a potent and selective K 1.1 inhibitor overcame the chemoresistance of the MG-63 and human chondrosarcoma SW-1353 spheroid models to paclitaxel, doxorubicin, and cisplatin. Among several multidrug resistance ATP-binding cassette transporters, the expression of the multidrug resistance-associated protein MRP1 was upregulated in both spheroids and restored by the inhibition of K 1.1. Therefore, the pharmacological inhibition of K 1.1 may be an attractive new strategy for acquiring resistance to chemotherapeutic drugs in the TME of K 1.1-positive sarcomas.