The fatigue strength and failure behavior of A5754-O adhesively bonded single lap joints by a hot-curing epoxy adhesive were investigated in this paper. The single lap joints tested include balanced substrate joints (meaning same thickness) and unbalanced substrate joints, involving combinations of different substrate thicknesses. Cyclic fatigue test results show that the fatigue strength of bonded joints increase with the increasing substrate thickness. SEM and Energy Dispersive X-ray (EDX) were employed to investigate the failure mode of the joints. Two fatigue failure modes, substrate failure and failure within the adhesive were found in the testing. The failure mode of the joint changes from cohesive failure to substrate failure as the axial load is decreased, which reveals a fatigue resistance competition between the adhesive layer and the aluminum substrate.