Acacetin, a natural flavonoid compound, has been proven to exert anti-inflammatory and immunomodulatory effects. Kv1. 3 channels, highly expressed in human T cells, are attractive therapeutic targets to treat inflammatory and immunological disorders. The present study was designed to characterize the inhibition of Kv1. 3 channels by Acacetin in human T cells and examine its role in T cell activation. Whole-cell patch-clamp was applied to record the Kv1. 3 and K Ca currents in human T cells; Western blot was used to detect Kv1. 3 expression as well as NFAT1 and NF-κB activity; Fluo-4, CCK-8 and an ELISA kit were used to measure Ca 2+ influx, proliferation, and IL-2 secretion, respectively. Acacetin decreased the Kv1. 3 current, accelerated the decay rate and negatively shifted the steady-state inactivation curves in a concentration-dependent manner. The IC 50 values at+ 40 mV for peak and the current at end of pulse were 21.09±2.75 and 3.63±0.25 µmol/L, respectively. Treatment with Acacetin for 24 h significantly inhibited Kv1. 3 protein expression. Additionally, paralleling Kv1. 3 inhibition, Acacetin also inhibited Ca 2+ influx, the Ca 2+-activated transcription factors NFAT1, NF-κB p65/p50 activity, and proliferation as well as IL-2 production. Small interfering RNA against Kv1. 3 reduced the inhibitory effect of Acacetin on IL-2 secretion. Acacetin blocks the Kv1. 3 channel and inhibits human T cell activation. This action most likely contributes to its immunomodulatory and anti-inflammatory actions.