α-secretase and β-secretase are known to compete for amyloid precursor protein (APP) processing and thus play a vital role in Alzheimer’s disease pathogenesis. A disintegrin and metalloproteinase 10 (ADAM10) and β-site APP cleaving enzyme 1 (BACE1) mediate the major activities of α-secretase and β-secretase in brain and share various common substrates. However, whether they function separately or together is poorly understood. Here, we show that ADAM10 and BACE1 co-localize in the neurites of mouse primary neurons. Co-immunoprecipitation and fluorescence resonance energy transfer analysis revealed that ADAM10 and BACE1 interact with each other under both endogenous and exogenous conditions. In addition, we found that ADAM10 enhances the proteolysis of neural cell adhesion molecule close homolog of L1 (CHL1) by BACE1. Further studies found that ADAM10–BACE1 interaction interfering peptide LT52 attenuates the regulation of ADAM10 on BACE1-mediated cleavage of CHL1. Our data indicate that ADAM10–BACE1 interaction regulates the proteolysis of some specific substrates and may play a potential role in brain function.