Non-typhoidal Salmonella (NTS) serovars are a leading cause of foodborne disease globally. Generalist serovars like S.Typhimurium often cause gastroenteritis in humans and farmed animals while invasive NTS serovars cause bacteraemia in a limited number of hosts, such as S.Dublin and S.Choleraesuis, which cause typhoidal disease in cattle and pigs, respectively, and occasionally in humans. Acquired via the faecal-oral route, all serovars traverse through the intestines where they encounter a decreasing gradient of bile from the duodenum to the distal ileum, where bile is reabsorbed. Previous research showed that while S.Typhimurium downregulates Salmonella pathogenicity island 1 (SPI-1) and motility genes in response to bile, S.Typhi, a strictly human typhoidal serovar, upregulates them. As invasive NTS serovars cause typhoidal disease in their respective hosts, we were keen to understand how they respond to host-specific bile. We cultured serovar-representative strains of S.Typhimurium (ST4/74), S.Dublin (SD3246) and S.Choleraesuis (SCA50) in the presence and absence of bile from cattle and pigs and performed RNA sequencing. We found that, with and without bile, ST4/74 expressed flagellin genes at higher levels than SD3246 and SCSA50, consistent with greater inflammation observed in vivo, and SPI-1 gene expression was higher in SD3246 and SCSA50, consistent with their greater in vivo invasiveness. However, a significant reduction in genome-wide transcription was observed in the presence of bile suggesting that NTS serovars may use bile as a spatial cue in vivo by downregulating expression of virulence factors in the intestines until arriving at the distal ileum, the preferential site of colonisation.