A novel dissimilar resistance spot welding of Ti6Al4V alloy and 316L stainless steel was performed via copper as interlayer by using optimal electrodes. A significant improvement in nugget morphology being of less defects was achieved with increasing the copper interlayer thickness from 50 μm to 400 μm. Thinner interfacial reaction layers at both the nugget/titanium interface and the nugget/stainless steel interface and finer equiaxed dendritic grained structure at the inner nugget were formed with copper interlayer thickness of 300 μm compared with those without interlayer. The formation of TiCu and Ti2Cu was facilitated with the incorporation of Cu into the nugget, which effectively inhibited the formation of the brittle Ti–Fe intermetallic compounds including Fe2Ti. The nugget in the welded joint with copper interlayer in 300 μm thickness exhibited a far lower microhardness than that of the interlayer-free welded joint, and the average microhardness reduced to 604 HV in the inner nugget, which was about 36% lower than that of the interlayer-free welded joint. The tensile shear load of the welded joint exhibited an increased tendency with increasing interlayer thicknesses from 50 μm to 300 μm, and the maximum tensile shear load of 6.3 kN was obtained with the interlayer thickness of 300 μm, which was ∼97% higher than that of the interlayer-free welded joint. The welded joint with copper interlayer exhibited hybrid ductile and fragile fractured characteristics. The work would provide a facile and cost-effective method to achieve a reliable welded joint of Ti alloy and stainless steel.