Flagella are the main motility structure of that affects the adhesion, colonization, and virulence of in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein () on the phenotype of . The gene deletion mutant (Δ) and its corresponding complementary strains (: : ) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The Δ mutant and the : : complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, Δ, and : : , the results showed that the growth rate and maximum biomass of Δ mutant decreased than that of CD630. The Δ mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : strain. Moreover, the motility was significantly reduced in the Δ mutant. Interestingly, the motility of the : : strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the Δ mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of Δ mutant reduced considerably compared to the CD630 strain and recovered in the : : strain. We conclude that the deletion of the gene significantly reduced the swimming motility of , suggesting that the gene is essential for the motility of . The gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of . These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of .