ABSTRACTGroup BStreptococcus(GBS) can cause many serious infections and result in severe symptoms depending on the infected organs. To survive and initiate infection from the gastrointestinal tract, GBS must resist physiochemical factors, such as bile salts, a potent antibacterial compound in the intestine. We found that GBS isolated from diverse sources all possess the capability to defend bile salts and permit survival. By constructing the GBS A909 transposon mutant library (A909Tn), we identified several candidate genes that might participate in the bile salt resistance of GBS. TherodAandcsbDgenes were validated as relevant to bile salt resistance. TherodAgene was anticipated to be related to peptidoglycan synthesis and influence the bile salt resistance of GBS by cell wall construction. Notably, we found that thecsbDgene worked as a bile salt resistance response factor and influenced several ABC transporter genes, specifically at the later growth period of GBS under bile salt stress. We further detected the marked intracellular bile salt accumulation in ΔcsbDby hydrophilic interaction chromatography-liquid chromatography/mass spectrometry (HILIC-LC/MS). Collectively, we showed a novel GBS stress response factor,csbD, contributes to bacterial survival in bile salts by sensing bile salt stress and subsequently induces transcription of transporter genes to excrete bile salts.IMPORTANCEGBS, a conditional pathogenetic colonizer of the human intestinal flora, can cause severe infectious diseases in immunocompromised patients. Therefore, it is critical to understand the factors that contribute to the resistance to bile salts, which are abundant in the intestine but harmful to bacteria. We identifiedrodAandcsbDgenes involved in bile salt resistance using a transposon insertion site sequencing (TIS-seq) based screen. TherodAgene products might be involved in peptidoglycan synthesis as important contributors to stress resistance including bile salts. However, thecsbDgene conferred bile salt resistance by promoting transporter genes transcription at the later growth period of GBS in response to bile salts. These findings developed a better understanding of the stress response factorcsbDon the bile salt resistance of GBS.