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In this paper, the assessment of the electrical and mechanical performance of the Cu nanowire anisotropic conductive film (NW-ACF) flip-chip interconnects has been performed. The influence of bonding conditions (bonding temperature and bonding force) has been studied in order to understand their impact on bonding resistance and shear strength for two types of NW-ACF based on different templates with varying porosity and thickness. An overgrowth-stripping method has been employed to obtain uniform NW (200 and 220 nm) arrays in the track-etch polymer membranes. The bonding substrate was coated with indium so that a diffusion soldering between Cu NWs and bulk In film can occur at a relatively low eutectic point (156.6°C). The bonding interface and the fractural surface of NW-ACFs have been characterized by scanning electron microscope and energy dispersive X-ray analysis. Under optimized fabrication conditions, process at the bonding profile of 200°C, 20 N shows that the z-axis resistance can be below 1 mΩ and the shear strength can be above 10 MPa, which is very promising for the fine pitch, 3-D interconnection applications in the future.