Genetic variabilities cause variations in the efficiency of most vaccines. Variations in gut microbiota composition may be also implied. We studied 4 experimental or commercial lines of laying hens. A strong disruption of the gut microbiota was induced by a cocktail of antibiotics given at two different times. Vaccine response was monitored by hemagglutination inhibition assays and the composition of the cecal microbiota was assessed by the analysis of amplified 16S rRNA gene sequences. The vaccine response level differed according to the line after vaccination. The effect of antibiotics on the vaccine response varied according to the line, with different dynamics over time. Finally, antibiotics intake impacted the composition of the caecal microbiota at the end of the experiment for each line. This experiment confirms the effect of both gut microbiota and genetic lineage on the level of vaccine response.