The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health crisis. The nucleocapsid (N) protein plays a pivotal role in a variety of biological processes in the life cycle of SARS-CoV-2, such as viral assembly. In this study, we investigated the liquid-liquid phase separation (LLPS) capacity of the N protein of seven SARS-CoV-2 strains, including the variants of concern (VOC) and interest (VOI), and its impact on viral replication. Using bioinformatic tools, we analyzed 11,433,558 complete genomes of SARS-CoV-2 and revealed a high degree of sequence conservation of N gene. While all the seven N proteins could undergo LLPS with RNA, the mutations in N impair its capacity of LLPS. With a SARS-CoV-2 trans-complementation system, we showed that SARS-CoV-2 variants carrying mutated N proteins exhibit impaired replication, highlighting the importance of LLPS of N in viral replication. We further demonstrated that (-)-gallocatechin gallate (GCG) efficiently inhibits the LLPS of N proteins and significantly suppresses the replication of different SARS-CoV-2 strains. Thus, our findings indicate that targeting the N-LLPS could be a viable strategy for the development of antiviral treatments against various SARS-CoV-2 strains, including those yet to emerge.