We have fabricated a junction consisting of an α-axis oriented film and a (001) substrate of YBa2Cu3Ox. First, a multi-layer structure has been prepared by film deposition at rather low substrate temperature, and the junction with an area of 40×40 μm2 has been fabricated by conventional photolithography and etching technique. This junction exhibits supercurrent below 65 K and Shapiro-steps under microwave irradiation at 5 GHz. This Josephson junction is the first one formed on the high-Tc single crystal substrate, which has high possibility of improvement in reproducibility due to the high quality electrodes.