A Mixed-voltage input and output (I/O) buffer including a pre-driver unit, a bulk-voltage generating unit, a first to a third transistors and an input stage unit is provided. The pre-driver unit outputs a first source/drain and a second signal. The bulk-voltage generating unit determines whether a first voltage or a pad voltage is used as a bulk voltage according to the pad voltage level. A gate of the first transistor receives the first signal, and a bulk, a first source/drain and a second source/drain of the first transistor are respectively coupled to the bulk voltage, the first voltage and the pad. A gate of the third transistor receives the second signal, and a first source/drain and a second source/drain of the third transistor are respectively coupled to the input stage unit for receiving an input signal from the pad and a second voltage.