The ubiquitin ligase Nedd4-like (Nedd4L, or Nedd4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out Nedd4L in mice intestinal epithelium (Nedd4L(f/f);Vil-Cre(ERT2)) we show here that Nedd4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl-, suggesting that Na+/K+/2Cl(-) (NKCC1/SLC12A2) co-transporter and ENaC are targets of Nedd4L in the colon. In accordance, NKCC1 (and ENaC) protein abundance in the colon of the Nedd4L knock-out animals was increased, indicating that Nedd4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between Nedd4L and NKCC1, suggesting that Nedd4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in and (but not ) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for Nedd4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.