Surface condition, especially surface roughness of substrates, critically influences the adhesion of cold-sprayed titanium alloy coatings. To study this, Ti6Al4V (Ti64) coatings were deposited on Ti64 substrates with increasing surface roughness (Ra) from 0.05 µm (polished surface) to 5.4 µm (water-jet cut surface). It was found that the substrate surface roughness did not significantly affect the porosity, hardness and coating surface roughness because these properties were dependent on the deposition parameters such as propellant gas pressure and temperature and nozzle traverse speed. The adhesion test results showed that smoother substrate surfaces improved the coating bond strength of the cold-sprayed Ti64 coatings from about 7.1 MPa (Ra: 5.4 µm, interface failure) to 68.7 MPa (Ra: 0.05 µm, glue failure). The fracture characteristics of the debonded coating/substrate interfaces revealed that there were more adiabatic shear-induced craters observed on the smoother substrate surfaces. Finite element modeling also showed that the substrate surface features (i.e., peaks and valleys) possibly prevented the intimate contact between the particles and substrate and thus induced the non-uniform distributions of temperature, stress and strain at the particle/substrate interface.