Epithelial sodium channels (ENaCs) have been reported to affect cancer cell development, including the processes of proliferation, apoptosis, and cell motility. Previously, we have shown the stimulating effect of extracellular serine protease trypsin on the activity of amiloride-insensitive ENaC-like channels in human leukemia K562 cells, where the expression of all ENaC subunits was found. However, the effect of serine proteases of various specificities on sodium channels and its functional role in the regulation of cancer progression in transformed blood cells remains unclear. In this study, using a single-channel whole-cell patch-clamp approach, we established that serine proteases α-chymotrypsin and plasmin directly activate sodium channels in leukemia K562 and U937 cell lines. δ-ENaC knockdown in K562 cells prevents the activation of the channels by α-chymotrypsin, confirming the involvement of δ-ENaC in protease-induced channels formation. Using an migration assay, we have shown for the first time that α-chymotrypsin and plasmin significantly reduced migration and invasion rate of K562 cells; δ-ENaC KD partially abolished this effect. The same effect of α-chymotrypsin was confirmed on the migration of different human leukemia cell lines (U937, HL-60). Incubation of the cells with serine proteases in the presence of their inhibitors (SBTI or α-antiplasmin) didn't affect cell migration/invasion, indicating an important role of their proteolytic activity. Moreover, there was no effect of α-chymotrypsin on the migration of MOLT-4 leukemia cells, which do not express functionally active ENaCs. Our data imply that extracellular serine proteases, as universal regulators of sodium permeability via ENaC, decrease leukemia cells migration.