In this study, we examine whether an anti‐inflammatory thiourea derivative, compound #326, actions on ion channels. The effects of compound #326 on Ca2+‐activated K+ channels were evaluated by patch‐clamp recordings obtained in cell‐attached, inside‐out or whole‐cell configuration. In pituitary GH3 cells, compound #326 increased the amplitude of Ca2+‐activated K+ currents (IK(Ca)) with an EC50 value of 11.6 μM, which was reversed by verruculogen, but not tolbutamide or TRAM‐34. Under inside‐out configuration, a bath application of compound #326 raised the probability of large‐conductance Ca2+‐activated K+ (BKCa) channels. The activation curve of BKCa channels was shifted to less depolarised potential with no modification of the gating charge of the curve; consequently, the difference of free energy was reduced in the presence of this compound. Compound #326‐stimulated activity of BKCa channels is explained by a shortening of mean closed time, despite its inability to alter single‐channel conductance. Neither delayed‐rectifier nor erg‐mediated K+ currents was modified. Compound #326 decreased the peak amplitude of voltage‐gated Na+ current with no clear change in the overall current–voltage relationship of this current. In HEK293T cells expressing α‐hSlo, compound #326 enhanced BKCa channels effectively. Intriguingly, the inhibitory actions of compound #326 on interleukin 1β in lipopolysaccharide‐activated microglia were significantly reversed by verruculogen, whereas BKCa channel inhibitors suppressed the expressions of inducible nitric oxide synthase. The BKCa channels could be an important target for compound #326 if similar in vivo results occur, and the multi‐functionality of BKCa channels in modulating microglial immunity merit further investigation. Compound #326‐stimulated activity of BKCa channels is explained by a shortening of mean closed time, despite its inability to alter single‐channel conductance. Intriguingly, the inhibitory actions of compound #326 on interleukin 1β in lipopolysaccharide‐activated microglia were significantly reversed by verruculogen, whereas BKCa channel inhibitors suppressed the expressions of inducible nitric oxide synthase.