This experiment was conducted to study the effects of drinking water supplementation of synbiotic product PoultryStar®sol (containing Lactobacillus reuteri, Bifidobacterium animalis, Pediococcus acidilactici, Enterococcus faecium, and fructo-oligosaccharide) in laying hens with and without a Salmonella challenge. A total of 384 one-day-old layer chicks were randomly distributed to the drinking water synbiotic supplementation or control groups. At 14 wk of age, the birds were vaccinated with a Salmonella vaccine, resulting in a 2 (control and synbiotic) X 2 (non-vaccinated and vaccinated) factorial arrangement. At 24 wk of age, half of the birds in the vaccinated groups and all the birds that were not vaccinated were challenged with Salmonella Enterica serotype Enteritidis, resulting in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial arrangment. At 8 d post-Salmonella challenge, synbiotic supplementation decreased (P = 0.04) cecal S. Enteritidis in the challenge group compared to the un-supplemented challenge group. Birds that were supplemented with synbiotic in the vaccine + challenge group had significantly greater cecal B. animalis and P. acidilactici percentage at 10 d post-Salmonella challenge than the birds in the vaccine + challenge group without synbiotic supplementation. At 3 d post-Salmonella challenge, birds that were supplemented with synbiotic in the challenge group had significantly greater cecal L. reuteri percentage than the birds in the challenge group without synbiotic supplementation. At 17 d post-Salmonella challenge, synbiotic supplementation increased bile anti-Salmonella IgA in the challenge group compared to the birds in the challenge group without synbiotic supplementation by 76.0%. At 10 d (P < 0.01) and 30 d (P = 0.05) post-Salmonella challenge, synbiotic supplementation decreased LITAF mRNA expression compared to the un-supplemented groups. At 3 d post-Salmonella challenge, synbiotic supplementation in the vaccine group had longer jejunal villi compared to the vaccine group without synbiotic supplementation. This experiment demonstrated that drinking water supplementation of the synbiotic product evaluated can significantly manipulate immune response and intestinal microbiota of laying hens post-Salmonella challenge to handle the challenge effectively.