Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile capable of catalyzing the oxidation of ferrous iron and sulfur reducing compounds. During the dissolution of ores A. ferrooxidans adheres to the solid substrate by hydrophobic and hydrophilic interactions and also extracellular polymeric substances may be involved. Nevertheless, many of these agents that participate in the process are unknown. Therefore, the identification and characterization of the extracellular proteome of A. ferrooxidans was addressed during this work. First, by searching the genome of A. ferrooxidans ATCC 23270 the putative genes coding for proteins present in most of the different secretion systems (type I, II, IV, V systems and the Usher pathway) were found. Second, when the secretion signal prediction server SubCel 1.0 was used, 26.7 % of the total open reading frames showed possible signals that would allow these proteins to leave the cytoplasm towards the internal and outer membranes, the periplasm, or the extracellular milieu. By using 2D-NEPHGE and MS sequencing we identified the eleven most abundant proteins present in the extracellular fraction. Proteins related to the transport and binding of solutes and the folding of proteins, among others were identified. Along with the non-classical pathway, the protein secretion systems identified could constitute the secretion machinery for the proteins found in the extracellular sub-proteome of A. ferrooxidans that as a whole constitute its secretome. Further studies of the extracellular proteins from this biomining microorganism will be important to find out their possible role if any, during bacteria-mineral interactions.