Epithelial Na+ channels (ENaCs) mediate sodium reabsorption in the cortical collecting duct (CCD), but the regulatory pathways that modulate the activity of these channels are incompletely understood. Here, we observed that endothelin-1 (ET-1) attenuates ENaC activity acutely by reducing the channel's open probability and chronically by decreasing the number of channels in the plasma membrane. To investigate whether beta1Pix, a signaling protein activated by ET-1, mediates ENaC activity, we reconstituted ENaC in CHO cells with or without coexpressed beta1Pix and found that beta1Pix negatively regulates ENaC. Knockdown of betaPix in native principal cells abolished the ET-1-induced decrease in ENaC channel number. Furthermore, we found that betaPix does not decrease ENaC activity through its guanine nucleotide exchange factor (GEF) activity for Rac1 and Cdc42. Instead, coexpression of beta1Pix mutant constructs revealed that beta1Pix affects ENaC activity through binding 14-3-3 proteins. Coimmunoprecipitation experiments supported a physical interaction between beta1Pix and 14-3-3beta in cultured principal cells. Coexpression of 14-3-3beta increased ENaC activity in CHO cells, but concomitant expression of beta1Pix attenuated this increase. Recruitment of 14-3-3beta by beta1Pix impaired the interaction of 14-3-3beta with the ubiquitin ligase Nedd4-2, thereby promoting ubiquitination and degradation of ENaC. Taken together, these results suggest that the inhibitory effects of chronic ET-1 on ENaC result from betaPix interacting with the 14-3-3/Nedd4-2 pathway.