A disintegrin and metalloprotease-8 (ADAM8) is thought to play a role in cancer and inflammatory diseases such as allergy, arthritis, and asthma. Despite the implication of ADAM8 in these diseases, the functional role of ADAM8 catalytic activity remains unclear. In this report, we demonstrate that an early critical autolytic event, we have termed pre-processing, is accelerated at acidic pH (pH 5.5) while autolytic activation is abrogated under the same conditions. Likewise, we found that pre-processing is hindered and autolytic activation is facilitated in neutral pH conditions, and thus demonstrates a pH-dependent shift in substrate selectivity. This finding is further supported by two peptide substrates corresponding to the pre-processing and C-terminal scissile bonds that were preferentially cleaved at acidic and neutral pH, respectively. Lastly, we found fibronectin cleavage to be attenuated at pH 5.5, while two novel substrates, brevican, and vitronectin, were readily cleaved in neutral or acidic conditions.