Both alternative sigma factor RpoS and ubiquitous secondary messenger c-di-GMP participate in the biofilm forming of Salmonella Pullorum; however, the relationship between RpoS and c-di-GMP-regulated genes during biofilm forming remains unclear. In this study, nine genes related with c-di-GMP regulation were found to be differentially expressed (P<0.01) by RNA-seq analysis when compared with S. Pullorum strain S9 and its rpoS deletion strain S9S. Specifically, the rpoS deletion strain S9S had higher transcription level of gene STM1703 and lower c-di-GMP concentration and biofilm-forming ability than S9 (P<0.01). The STM1703 gene deletion in strains S9 and S9S significantly enhanced the c-di-GMP concentration and the biofilm-forming ability (P<0.01). qRT–PCR analysis showed that rpoS deletion or P193L substitution in RpoS increased the transcription level of the STM1703 gene by decreasing the transcription levels of the csrA and STM1344 genes (P<0.01). Overall, RpoS-mediated STM1703 negatively regulates the biofilm formation of S. Pullorum by degrading c-di-GMP.