Aims: The primary goal of this study was to characterize the existence of a functional c-di-GMP pathway in the bioleaching bacterium Acidithiobacillus ferrooxidans. Methods and Results: A bioinformatic search revealed that the genome sequence of At. ferrooxidans ATCC 23270 codes for several proteins involved in the c-di-GMP pathway, including diguanylate cyclases (DGC), phosphodiesterases and PilZ effector proteins. Overexpression in Escherichia coli demonstrated that four At. ferrooxidans genes code for proteins containing GGDEF/ EAL domains with functional DGC activity. MS/ MS analysis allowed the identification of c-di-GMP in nucleotide preparations obtained from At. ferrooxidans cells. In addition, c-di-GMP levels in cells grown on the surface of solid energetic substrates such as sulfur prills or pyrite were higher than those measured in ferrous iron planktonic cells. Conclusions: At. ferrooxidans possesses a functional c-di-GMP pathway that could play a key role in At. ferrooxidans biofilm formation during bioleaching processes. Significance and Impact of the Study: This is the first global study about the c-di-GMP pathway in an acidophilic bacterium of great interest for the biomining industry. It opens a new way to explore the regulation of biofilm formation by biomining micro-organisms during the bioleaching process.