Pannexin 1 (Panx1), a member of the second gap junction protein family identified in vertebrates, appears to preferentially form non-junctional membrane channels. A candidate regulatory protein of Panx1 is the potassium channel subunit Kvbeta3, previously identified by bacterial two-hybrid strategies. Here, we report on the physical association of Panx1 with Kvbeta3 by immunoprecipitation when co-expressed in a neuroblastoma cell line (Neuro2A). Furthermore, in vivo co-expression of Panx1 and Kvbeta3 was shown to occur in murine hippocampus and cerebellum. Kvbeta3 is known to accelerate inactivation of otherwise slowly inactivating potassium channels under reducing conditions. We subsequently found that Panx1 channel currents exhibit a significant reduction when exposed to reducing agents, and that this effect is attenuated in the presence of Kvbeta3. Apparently, Kvbeta3 is involved in regulating the susceptibility of Panx1 channels to redox potential. Furthermore, the Panx1 channel blockers carbenoxolone and Probenecid were less effective in inhibiting Panx1 currents when Kvbeta3 was co-expressed. The influence of Kvbeta3 on Panx1 is the first example of modulation of Panx1 channel function(s) by interacting proteins, and suggests the physiological importance of sensing changes in redox potentials.