The Preiss-Handler pathway, which salvages nicotinate (NA) for NAD synthesis, is an indispensable biochemical pathway in land plants. Various NA conjugations (mainly methylation and glycosylation) have been detected, and have long been proposed for NA detoxification in plants. Previously, we demonstrated that NA O-glucosylation functions as a mobilizable storage form for NAD biosynthesis in the Brassicaceae. However, little is known about the functions of other NA conjugations in plants. In this study, we firstly found that N-methylnicotinate is a ubiquitous NA conjugation in land plants. Further, we functionally identified a novel methyltransferase (At3g53140, NANMT), which is mainly responsible for N-methylnicotinate formation, from Arabidopsis. We also established that trigonelline (Tg) is a detoxification form of endogenous NA in plants. Combined phylogenetic analysis and enzymatic assays revealed that NA N-methylation activity was likely derived from duplication and subfunctionalization of an ancestral caffeic acid O-methyltransferase (COMT) gene in the course of land plant evolution. COMT enzymes, which function in S-lignin biosynthesis, also have weak NANMT activity. Our data suggest that NA detoxification conferred by NANMT and COMT might have facilitated the retention of the Preiss-Handler pathway in land plants.