The Arabidopsis thaliana NONEXPRESSER OF PR GENES1 (NPR1, also known as NIM1) protein is an essential positive regulator of salicylic acid (SA)-induced PATHOGENESIS-RELATED (PR) gene expression and systemic acquired resistance (SAR). PR gene activity is regulated at the level of redox-dependent nuclear transport of NPR1. NPR1 interacts with members of the TGA family of transcription factors that are known to bind to SA-responsive elements in the PR-1 promoter. In an attempt to identify proteins involved in SA-mediated signal transduction, we previously described the isolation of three novel genes encoding distinct albeit structurally related proteins designated NIMIN1 (for NIM1-INTERACTING1), NIMIN2, and NIMIN3 that interact with NPR1 in the yeast two-hybrid system. Here, we show that NIMIN1 and NPR1 can be copurified from plant extracts, providing biochemical evidence for their interaction. We provide functional evidence for this interaction by describing transgenic plants constitutively expressing high amounts of NIMIN1. These plants show reduced SA-mediated PR gene induction and a compromised SAR, thus mimicking the described phenotype conferred by npr1. Moreover, they showed reduced RESISTANCE gene-mediated protection. These effects were dependent on the ability of NIMIN1 to interact with NPR1. Mutant plants with a T-DNA insertion in NIMIN1 as well as transgenic plants with reduced NIMIN1 mRNA levels showed hyperactivation of PR-1 gene expression after SA treatment but no effect on the disease resistance phenotype. Our results strongly suggest that NIMIN1 negatively regulates distinct functions of NPR1, providing a mechanism to modulate specific features of SAR.