The serine proteases CAP1/Prss8 and CAP3/St14 are identified as ENaC channel-activating proteases in vitro, highly suggesting that they are required for proteolytic activation of ENaC in vivo. The present study tested whether CAP3/St14 is relevant for renal proteolytic ENaC activation and affects ENaC-mediated Na+ absorption following Na+ deprivation conditions. CAP3/St14 knockout mice exhibit a significant decrease in CAP1/Prss8 protein expression with altered ENaC subunit and decreased pNCC protein abundances but overall maintain sodium balance. RNAscope-based analyses reveal co-expression of CAP3/St14 and CAP1/Prss8 with alpha ENaC in distal tubules of the cortex from wild-type mice. Double CAP1/Prss8; CAP3/St14-deficiency maintained Na+ and K+ balance on a Na+-deprived diet, restored ENaC subunit protein abundances but showed reduced NCC activity under Na+ deprivation. Overall, our data clearly show that CAP3/St14 is not required for direct proteolytic activation of ENaC but for its protein abundance. Our study reveals a complex regulation of ENaC by these serine proteases on the expression level rather than on its proteolytic activation.