Antibiotic resistance genes are usually tightly controlled by transcription factors and RNA regulatory elements including sRNAs, riboswitches, and attenuators, and their expression is activated to respond to antibiotic exposure. In previous work, we revealed that the rppA gene is regulated by attenuator LRR and two mistranslation products in Bacillus thuringiensis BMB171. However, its function and promoter regulation is still not precise. In this study, we demonstrated that the encoding product of the rppA gene acts as an ARE1 ABC-F protein and confers resistance to antibiotics virginiamycin M1 and lincomycin when overexpressed. Besides the reported attenuator LRR, the expression of the rppA gene is controlled by the sigma factor SigA and a global transcription factor CcpA. Consequently, its promoter activity is mainly maintained at the stationary phase of cell growth and inhibited in the presence of glucose. Our study revealed the function and regulation of the rppA gene in detail. KEY POINTS: • The RppA protein acts as an ARE1 ABC-F protein • The rppA gene confers resistance to antibiotics virginiamycin M1 and lincomycin when overexpressed • The expression of the rppA gene is regulated by the sigma factor SigA and the pleiotropic regulator CcpA.