Setaria viridis is an emerging model system for the genetic and molecular characterization of cereals and bioenergy crops. Here, we describe a detailed procedure for genetic transformation of the S. viridis accession line ME034V-1. This method utilizes callus generated from mature seeds for infection with Agrobacterium tumefaciens strain AGL1 to regenerate hygromycin-resistant stable transgenic plants. It takes approximately 7 weeks to generate callus from mature seeds, 11-17 weeks from infection to the regeneration of transgenic lines, and an additional 3-4 weeks for plant growth in the greenhouse for seed collection. The protocol as presented consistently results in transformation frequency of approximately 25% for the generation of transgenic plants, with fewer escapes and higher survivability in soil for optimal seed collection. © 2021 Donald Danforth Plant Science Center. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of S. viridis (Accession ME034V-1) callus from mature seeds Basic Protocol 2: Agrobacterium-mediated transformation of callus to generate transgenic plants Basic Protocol 3: Plantlet transplantation in soil, plant growth in greenhouse, and seed collection Support Protocol: Preparation of Agrobacterium culture for infection.