Chronic infection of HBV is a serious global health issue. However, the HBV covalently closed circular DNA (cccDNA) cannot be completely cleaned in liver. Here, we engineer a novel polypeptide, termed D-TTK001, targeting HBV X protein (HBx) against HBV cccDNA. D-TTK001 is a D-type polypeptide, which has a high binding affinity to HBx. Interestingly, it is able to against HBV efficiently in primary human hepatocytes (PHHs) from three donors. D-TTK001 displays high activities against HBV, remarkably reducing the levels of cccDNA in the HBV-infected human liver-chimeric mice. Mechanically, D-TTK001 depresses cccDNA transcription by regulating the epigenetic modification of cccDNA minichromosome and disturbing the interaction of HBx with DDB1 in the cells. D-TTK001 decreases the levels of cccDNA by down-regulating MSL2, leading to the increase of APOBEC3B in liver. When combined with entecavir or interferon-α, D-TTK001 shows additive antiviral effects. Clinically, D-TTK001 is a strong candidate to clear cccDNA against HBV.