Multicellular tumor three-dimensional spheroid models are spherical self-assembled aggregates of cancer cells. They are a valuable tool for studying the tumor microenvironment in solid tumors and are also important for investigating the characteristics of cancer stem cells such as chemoresistance. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, and decreased the protein degradation of the Ca²⁺-activated K⁺ channel K_Ca1.1 by down-regulating the E3 ubiquitin ligase, FBXW7. K_Ca1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of K_Ca1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation, and reversed by K_Ca1.1 inhibition. The inhibition of the E3 ubiquitin ligase, MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by K_Ca1.1 inhibition. Additionally, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by K_Ca1.1 inhibition. Collectively, the present results suggest the potential of K_Ca1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells. These suggest that the potential of K_Ca1.1 inhibitors as an effective therapeutic intervention in combination with antiandrogens.