Poultry vaccines are often administered using water as a diluent and applied using an oral or coarse spray method. Gel-based vaccine diluents are developed as an alternative vaccine delivery method. Gels are viscous, and gel droplets adhere more effectively to feathers giving the vaccine a longer time to be ingested (through preening). Data on the application of live bacterial vaccines using gel and its effect on gut colonization is limited. The present study tested a gel diluent prepared in various media, using a live, attenuated Salmonella Typhimurium vaccine, Vaxsafe ST. Gene expression of the vaccine in different dilutions was analyzed using the qPCR technique. Q-PCR and in vitro, results show that the use of nutrient-enriched diluents significantly enhances the viability or motility of the vaccine. The invasive capacity of the vaccine suspended in gel diluent was also tested in vitro. Results demonstrated that the vaccine suspended in gel diluent retained invasiveness. Day-old chicks were orally vaccinated with Vaxsafe ST suspended in gel diluent to characterize the in vivo colonization capacity of the vaccine. The results revealed that the VaxSafe ST suspended in gel diluent could efficiently colonize the caeca of chicks, which is required for the development of protective immunity.