This study specified the role of several key calcium-operating ion channels in contraction/relaxation of human detrusor muscle as possible target for overactive bladder (OAB) treatment. Detrusor samples, obtained from 18 males (average age 61.5 ± 5.9 years), were investigated by organ tissue bath method with following agents: diltiazem for L-type voltage-gated calcium channels; 3-fluropyridine-4-carboxylic acid (FPCA) for Orai-STIM channels; SKF 96365-hydrochloride for transient receptor potential (TRP) channels, T-type channels and Orai-STIM channels; 2- aminoethoxydiphenyl borate (2-APB) for inositol-triphosphate receptors (IP3Rs) and Orai-STIM channels. Oxybutynin and mirabegron were tested under the same conditions as controls. Mirabegron, 2-APB and FPCA exhibited the best suppressive effect on carbachol-induced detrusor contractility. As expressed by area under the contractile curve (AUCC), 2-APB, FPCA and mirabegron have similar AUCC: 1.79, 1.73, 1.73. The highest AUCC was 3.64 for diltiazem+SKF, followed by 3.21 for diltiazem, 3.16 for SKF and 2.94 for oxybutynin. The lowest median amplitude and contraction variability is for 2-APB followed by mirabegron and FPCA. There were significant differences between: 2-APB/FPCA vs.: ditiazem, diltiazem+SKF and SKF. Summary of results suggested the principal role of IP3Rs, Orai-STIM coupling and large-conductance calcium-activated potassium channels in detrusor contraction and pointed on Orai-STIM channels as possible targets for OAB pharmacotherapy.