Geminiviruses are single-stranded DNA viruses that infect a wide variety of plants and cause severe crop losses worldwide. The geminivirus replication initiator protein (Rep) binds to the viral replication origin and catalyzes DNA cleavage and ligation to initiate rolling circle replication. In this study, we found that the Tomato golden mosaic virus (TGMV) Rep is phosphorylated at serine-97 by sucrose nonfermenting 1-related protein kinase 1 (SnRK1), a master regulator of plant energy homeostasis and metabolism. Phosphorylation of Rep or the phosphomimic S97D mutation impaired Rep binding to viral DNA. A TGMV DNA-A replicon containing the Rep S97D mutation replicated less efficiently in tobacco (Nicotiana tabacum) protoplasts than in wild-type or Rep phosphorylation-deficient replicons. The TGMV Rep-S97D mutant also was less infectious than the wild-type virus in Nicotiana benthamiana and was unable to infect tomato (Solanum lycopersicum). Nearly all geminivirus Rep proteins have a serine residue at the position equivalent to TGMV Rep serine-97. SnRK1 phosphorylated the equivalent serines in the Rep proteins of Tomato mottle virus and Tomato yellow leaf curl virus and reduced DNA binding, suggesting that SnRK1 plays a key role in combating geminivirus infection. These results established that SnRK1 phosphorylates Rep and interferes with geminivirus replication and infection, underscoring the emerging role for SnRK1 in the host defense response against plant pathogens.,© 2018 American Society of Plant Biologists. All rights reserved.