The blocking effect of intracellular spermine (SPM) on the Kir2.1 channel is closely correlated with current flow, constituting one of the major bases of inward rectification. The flow dependence strongly suggests coupled movement of multiple ions in a single-file region of the pore. The location and the functional operation of this region, however, remain largely uncharacterized. Based on electrophysiological recordings in inside-out patches from Xenopus oocytes obtained by partial ovariectomy from frogs anesthetized with 0.1 % tricaine and pressure injected with wild-type or mutant Kir2.1 cRNA, we found that both the flux-coupling feature and the affinity of intracellular SPM block are decreased in the D172N but not S165L mutant channel. However, only the S165L mutant channel shows an evidently facilitated outward exit of the blocking SPM at strong depolarized potentials. On the other hand, the kinetics of SPM entry to and exit from the flow-dependent blocking site are markedly and correlatively slowed by specific E224 and E299 mutations, which always also disrupt the flux-coupling feature of SPM block. The slowed entry rates carry little voltage dependence, whereas the exit rates are always decelerated with more depolarization. Interestingly, the foregoing effect is present in the E299H mutant channel at pH 6.4 but not at pH 8.4, and the effect of E244/E299 mutations are mostly counteracted by concomitant mutations of R228/R260. Moreover, mutations involving residues I176 – A184 in the bundle crossing region markedly weaken the inward rectifying feature of SPM block of the Kir2.1 channel, and mutations of E224 and M183/A184 even alter the height of the same asymmetrical barrier to SPM permeation. Also, the channel seems to be able to accommodate at least two SPM molecules simultaneously, as the unblocking of the blocking SPM is accelerated in higher intracellular SPM concentration. We conclude that E224 and E299, probably through electrostatic interactions with R228 and R260, play a pivotal role in the “gating” of the bundle crossing region of the Kir2.1 channel pore. This region, which probably contains an outmost site involving D172 for SPM binding and is demarcated externally by a large energy barrier for SPM to cross at S165, is also responsible for the flux-coupling feature and the inward rectification property of the channel. In this regard, SPM intriguingly serves as both a flow-dependent pore blocker and a “gating particle” capable of opening the bundle crossing of the pore.