Abstract: Coxsackievirus B3 (CVB3) is the most common agent known to cause viral myocarditis. The viral genome encodes a single polyprotein that is cleaved to produce several proteins by virally encoded proteases. Most of this proteolytic processing is catalyzed by a cysteine protease called 3C. The 3C protease plays major role in viral replication and cellular damage. To understand the mechanism of 3C function in virus infected cells and also development of antiviral agants against the virus, a 3C expressing plasmid was constructed. The cDNA of 3C protease was synthesized using CVB3 infected cells through reverse transcription process and was cloned in pcDNA3.1- . The constructed plasmid was confirmed by sequencing and restriction enzyme analysis. By transfection of the constructed plasmid into HeLa and MCF-7 cells, we showed that 3C protease induced cell death through multiple converging pathways, such as down regulation of cellular factors and decreasing of mRNA transcripts. This affect on HeLa cells as stronger than MCF-7 cells.