Cholera, a diarrhoeal disease caused by Gram-negative bacterium Vibrio cholerae remains a global health threat in developing countries owing to its high transmissibility and increase in antibiotic resistance. The current issue is to overcome the problem of resistance by antimicrobial therapy. There is a pressing need for alternative strategies for treatment with an emphasis on anti-virulent approaches to alter the outcome of bacterial infections, especially given the rise in antimicrobial-resistant strains. Vibrio cholerae causes cholera by secreting virulence factors into the intestinal epithelial cells. Virulence factors help in cholera toxin production and colonization during infection. Here, we show that sodium butyrate (SB), a small molecule, had no effect on bacterial viability but was effective in suppressing the virulence attributes of V. cholerae. The production of cholera toxin (CT) was significantly reduced in a standard V. cholerae El Tor strain and two clinical isolates when grown in the presence of SB. Analysis of mRNA and protein levels further demonstrated that SB reduced the expression of the ToxT-dependent virulence genes like tcpA and ctxAB. DNA-protein interaction assays conducted at cellular (ChIP) and in vitro conditions (EMSA) indicated that SB weakens the binding between ToxT and its downstream promoter DNA, likely by blocking DNA binding. Furthermore, the efficacy of SB was confirmed by showing its anti-virulence activity in animal models. Collectively, these findings suggest that SB has the potential to be developed as an anti-virulence agent against V. cholerae in place of conventional antibiotics or as an adjunctive therapy to combat cholera.