The pyridine-based PNN ruthenium pincer complex (PNN)Ru(CO)(H) can catalyze the well-known dehydrogenative amidation reaction, but the mechanism is not fully understood. In this work, we find there exists an alternative metal-substrate cooperation mechanism in this reaction system, which is more favorable than the aromatization-dearomatization mechanism. The possible reaction of the excess base t-BuO- with catalyst species (PNN)Ru(CO)(H) is studied, indicating t-BuO- is able to facilitate the ligand substitution and enhance catalytic activities. With the bifunctional Ru-N moiety, the imine-substituted species (PN)(imine)Ru(CO)(H) 5 could serve as an alternative catalytic species and efficiently facilitate some elementary steps such as the hydrogen transfer, hydrogen elimination, and C-N coupling. Meanwhile, the C-N coupling step proceeds via the split of aldehydic C-H bond across the Ru(II)-imine bond, which results in an amide bond directly. The hemiaminal is uninvolved in the C-N coupling process. Finally, the formation of linear peptides and cyclic dipeptides are unveiled by the newly proposed mechanism. The metal-substrate cooperation could widely exist in transition metal catalyst systems with a large influence on the reaction activity.