Numerous reports have linked cytoskeleton associated proteins with the regulation of ENaC activity. The purpose of this study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here we show cytochalasin E treatment for 60 minutes can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show by single-channel patch clamp studies and measurements of short circuit current that ENaC activity, but not its density is altered by cytochalasin E induced disruption of the cytoskeleton. In non-treated cells 8 out of 33 patches (24%) had no measurable ENaC activity while in cytochalasin E treated cells 17 out of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in non-treated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 minutes was decreased compared to vehicle treated cells. The subcellular expression of fodrin changed significantly and several protein elements of the cytoskeleton decreased at least two fold after 60 minutes of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and MARCKS. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-MARCKS complex.