Although Lactobacillus species have been administered widely as probiotics in poultry production, the mechanisms responsible for their functionality are not well understood. The genetic tools available for use in lactobacilli are advanced but have not been applied widely to investigate their probiotic functionality in poultry. The genome sequence of Lactobacillus gallinarum ATCC 33199, originally isolated from the chicken crop, has recently been made available suggesting this organism as a potentially important model organism for probiotic research in poultry. In this study, we demonstrated the functionality of the pORI28 system for construction of isogenic knockout mutants in L. gallinarum ATCC 33199 using insertional inactivation of lacL as proof-of-principle. The establishment of an effective chromosomal integration system for use in L. gallinarum ATCC 33199 will provide a platform for functional genomic analyses to investigate the functionality of this model organism in the gastrointestinal tract of poultry.