A LuxI/R-like quorum sensing (QS) system (AfeI/R) has been reported in the acidophilic and chemoautotrophicAcidithiobacillusspp. However, the function of AfeI/R remains unclear because of the difficulties in the genetic manipulation of these bacteria. Here, we constructed differentafeImutants of the sulfur- and iron-oxidizerA.ferrooxidans, identified the N-acyl homoserine lactones (acyl-HSLs) synthesized by AfeI, and determined the regulatory effects of AfeI/R on genes expression, extracellular polymeric substance synthesis, energy metabolism, cell growth and population density ofA.ferrooxidansin different energy substrates. Acyl-HSLs-mediated distinct regulation strategies were employed to influence bacterial metabolism and cell growth ofA.ferrooxidanscultivated in either sulfur or ferrous iron. Based on these findings, an energy-substrate-dependent regulation mode of AfeI/R inA.ferrooxidanswas illuminated that AfeI/R could produce different types of acyl-HSLs and employ specific acyl-HSLs to regulate specific genes in response to different energy substrates. The discovery of the AfeI/R-mediated substrate-dependent regulatory mode expands our knowledge on the function of QS system in the chemoautotrophic sulfur- and ferrous iron-oxidizing bacteria, and provides new insights in understanding energy metabolism modulation, population control, bacteria-driven bioleaching process, and the coevolution between the acidophiles and their acidic habitats.