Objective · To uncover new transcriptional regulatorsscreening putative transcriptional regulators in Salmonella enterica serovar Typhimurium (S. Typhimurium) through genome informatics and molecular biology analyses. Methods · S. Typhimurium genome informatics was analyzed and 30 putative transcriptional regulators were screened out. All candidate genes were deletedλ-Red system. The log-phase acid tolerance response (ATR) ability was compared between knock-out (KO) strains and wild type (WT) strain. Cell model was used to detect the invasion ability and intracellular ability of KO strains that showed differences in acid stress compared to WT strain. The transcription level of phoP was detectedrealtime-PCR in the mutants involved in both ATR and cell infection. Results · All 30 deletion mutants were successfully constructed. ΔSTM14_0739, ΔSTM14_2717 and ΔSTM14_1646 showed increased log-phase ATR ability, while ATR abilities of ΔSTM14_4338, ΔSTM14_1965 and ΔSTM14_1878 decreased, compared with WT. ΔSTM14_0739 and ΔSTM14_2717 mutants showed weaker invasion ability in HeLa cells than WT, and ΔSTM14_1878 and ΔSTM14_2717 mutants showed stronger replication ability in RAW264.7 cells than WT. Realtime-PCR suggested STM14_2717 deletion had no effect on phoP transcription. Conclusion · This work discovers unknown transcriptional regulators, and provides clues for future research in S. Typhimurium pathogenesis.