Biofilm (bacterial biofilm, BF) are associated with most bacterial infections, which can help pathogens against adverse environment, such as antibiotic and phages. In order to exploid the mechanism involved with BF formation and anti-phage, six Salmonella paratyphi A (S. paratyphi A) mutants resistant to phage were analyzed in this study. Phages were dropped on bacteria line on LB plate containing Rif⁺(200 mg/L)to confirm phage resistance. Six mutants were cultured in 96 well plate with three replications to monitor biofilm formation and morphology. Site-directed mutagenesis and complementation test were used to confirm whether phage resistance was caused by gene mutant. Results of this study showed that all six mutants were resistant to phage, because those could growth with dropped 2.4×10⁹ phages and wild bacteria could not with dropped 1.2×10⁶ phages. Among six mutants, BF formation of mutant of σ-54-dependent translation regulator was significantly enhanced contrast with wild bacteria (P < 0.05, 1.1±0.2 vs 0.5±0.1). Bacteria aggregated to large irregular mass. Site-directed mutation of σ-54-dependent translation regulator with homologous recombination led to be resistant to phages. This resistance could be recovered by supplemented with plasmid expressed σ-54-dependent translation regulator. All those showed that σ-54-dependent translation of the regulator was associated with anti-phage and BF formation.