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In this paper, a novel anisotropic conductive film (ACF) flip chip bonding method using ultrasonic vibration for flip chip interconnection is demonstrated. The curing and bonding behaviors of ACFs by ultrasonic vibration were investigated using a 40-kHz ultrasonic bonder with longitudinal vibration. In situ temperature of the ACF layer during ultrasonic (U/S) bonding was measured to investigate the effects of substrate materials and substrate temperature. Curing of the ACFs by ultrasonic vibration was investigated by dynamic scanning calorimetry (DSC) analysis in comparison with isothermal curing. Die adhesion strength of U/S-bonded specimens was compared with that of thermo-compression (T/C) bonded specimens. The temperature of the ACF layer during U/S bonding was significantly affected by the type of substrate materials rather than by the substrate heating temperature. With room the temperature U/S bonding process, the temperature of the ACF layer increased up to 300degC within 2 s on FR-4 substrates and 250degC within 4 s on glass substrates. ACFs were fully cured within 3 s by ultrasonic vibration, because the ACF temperature exceeded 300degC within 3 s. Die adhesion strengths of U/S-bonded specimens were as high as those of T/C bonded specimens both on FR-4 and glass substrates. In summary, U/S bonding of ACF significantly reduces the ACF bonding times to several seconds, and also makes bonding possible at room temperature compared with T/C bonding which requires tens of seconds for bonding time and a bonding temperature of more than 180degC.