Ca²⁺-activated K⁺ channels play a critical role in the proliferation, apoptosis, migration, adhesion, and metastasis of various types of cancer cells by controlling cell volumes and Ca²⁺ signaling. The amplification of them correlated with a high tumor stage and poor prognosis, and has potential as tumor grade‐associated markers. The hypoxic tumor microenvironment (TME) promotes the anti-cancer drug resistance and stemness of solid tumors. Three‐dimensional (3D) in vitro cancer spheroid models mimic the TME of human solid tumors, and are an efficient tool for investigating chemoresistance and stemness. We here introduce the mechanisms underlying the post‐translational modification of K_Ca1.1 and the overcome of chemo- and antiandrogen-resistance by K_Ca1.1 inhibition in 3D cancer spheroid models. Cancer-associated immunosuppressive cells such as tumor-associated macrophages also promote tumorigenesis and anti-cancer drug resistance by secreting immunosuppressive cytokines/chemokines. We introduce the possible involvement of K_Ca3.1 in escape from anti-tumor immune surveillance by inhibiting immunosuppressive IL-10 and IL-8 expression in the differentiated M₂ macrophages.