The epithelial sodium channel (ENaC) is essential for regulating renal sodium excretion. Cleavage of the γENaC subunit increases ENaC activity both in vitro and in vivo; however, the involved endogenous proteases remain ambiguous. This study investigates the mechanistic roles of the pro-protein convertase (PCSK) family and the serine protease prostasin in proteolytic activation of ENaC. We used the M-1 mouse collecting duct cell line that natively expresses functional ENaC channels for pharmacological inhibition studies with pro-protein convertase inhibitor decanoyl-RVKR-chloromethyl ketone and serine protease inhibitor camostat mesilate. Knockout cell lines of prostasin and three PCSKs identified by mRNA expression were generated with the CRISPR/Cas9 system in M-1 cells. As an index of ENaC activity, the amiloride-sensitive short-circuit current (ISC) and transepithelial voltage (VTE) were measured on polarized cells on transwell filter supports in Ussing chambers. γENaC membrane expression and cleavage were tested by western blotting of surface biotinylated M-1 cells with an antibody against the C-terminal of γENaC. All values are means ± SE, compared with students t-test or ANOVA as appropriate. Pro-protein convertase inhibition decreased the amiloride-sensitive ISC in M-1 cells compared with controls (25.9±1.9 μA, n=5, vs. 45.7±7.0 μA, n=6, p<0.05). Similarly, serine protease inhibition decreased VTE (-34.5±7.8 mV, n=3, vs. -74.2±0.9 mV, n=3, p<0.05) but ISC was not significantly lower than controls (68.0±8.5 μA, n=3, vs. 74.0±2.3 μA, n=3, n.s.). Both inhibitors decreased membrane expression of γENaC, but only serine protease inhibition reduced γENaC cleavage (n=1). Knockout of the Pcsk3 (furin) gene completely abolished ENaC activity, while knockouts of the Pcsk4, Pcsk7, and Prss8 (prostasin) genes all showed decreased ENaC activity compared with wild-type M-1 cells. Knockout of PCSKs reduced γENaC surface expression, but only knockout of prostasin decreased γENaC cleavage (n=1). We propose that PCSKs are not directly involved in proteolytic ENaC activation. Both pharmacological inhibition and knockout of prostasin decreased ENaC activity and γENaC cleavage, thus suggesting a potential role in proteolytic ENaC activation.