Burkholderia seminalis strain TC3.4.2R3 is an endophytic bacterium isolated from sugarcane roots that produces antimicrobial compounds, facilitating its ability to act as a biocontrol agent against phytopathogenic bacteria. In this study, we investigated the thermoregulation of B. seminalis TC3.4.2R3 at 28 °C (environmental stimulus) and 37 °C (host-associated stimulus) at the transcriptional and phenotypic levels. The production of biofilms and exopolysaccharides such as capsular polysaccharides and the biocontrol of phytopathogenic fungi were enhanced at 28 °C. At 37 °C, several metabolic pathways were activated, particularly those implicated in energy production, stress responses and the biosynthesis of transporters. Motility, growth and virulence in the Galleria mellonella larvae infection model were more significant at 37 °C. Our data suggest that the regulation of capsule expression could be important in virulence against G. mellonella larvae at 37 °C. In contrast, B. seminalis TC3.4.2R3 failed to cause death in infected BALB/c mice, even at an infective dose of 107 CFU.mL-1. We conclude that temperature drives the regulation of gene expression in B. seminalis during its interactions with the environment.