The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or 2019 novel coronavirus (2019-nCoV), took tens of thousands of lives and caused tremendous economic losses. The main protease (Mpro) of SARS-CoV-2 is a potential target for treatment of COVID-19 due to its critical role in maturation of viral proteins and subsequent viral replication. Conceptually and technically, targeting therapy against Mpro is similar to target therapy to treat cancer. Previous studies show that GC376, a broad-spectrum dipeptidyl Mpro inhibitor, efficiently blocks the proliferation of many animal and human coronaviruses including SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), porcine epidemic diarrhea virus (PEDV), and feline infectious peritonitis virus (FIPV). Due to the conservation of structure and catalytic mechanism of coronavirus main protease, repurposition of GC376 against SARS-CoV-2 may be an effective way for the treatment of COVID-19 in humans. To validate this conjecture, the binding affinity and IC50 value of Mpro with GC376 was determined by isothermal titration calorimetry (ITC) and fluorescence resonance energy transfer (FRET) assay, respectively. The results showed that GC376 binds to SARS-CoV-2 Mpro tightly (KD = 1.6 μM) and efficiently inhibit its proteolytic activity (IC50 = 0.89 μM). We also elucidate the high-resolution structure of dimeric SARS-CoV-2 Mpro in complex with GC376. The cocrystal structure showed that GC376 and the catalytic Cys145 of Mpro covalently linked through forming a hemithioacetal group and releasing a sulfonic acid group. Because GC376 is already known as a broad-spectrum antiviral medication and successfully used in animal, it will be a suitable candidate for anti-COVID-19 treatment.