The nucleoid-associated protein Fis is a key transcriptional regulator in Gram-negative bacteria that supports rapid adaptation to environmental changes. In Yersinia pseudotuberculosis, Fis plays a critical, yet poorly understood role in virulence. Here, we present a comparative transcriptomic analysis of Y. pseudotuberculosis wild type and fis deletion mutant at environmental (25°C) and host-relevant (37°C) temperatures. Our data show that Fis modulates the expression of more than 600 genes acr