A novel coronavirus, SARS-CoV-2, caused a large respiratory outbreak in Wuhan, China in December 2019. This study shows that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating the virus. The results indicate that TMPRSS2 plays an important role in SARS-CoV-2 infection, similar to its role in SARS-CoV and MERS-CoV. Seven clinical specimens from SARS-CoV-2 infection cases were inoculated into VeroE6/TMPRSS2 cells, which were monitored for cytopathic effect (CPE). In five cases, clear CPE with cell detachment and syncytium formation was observed. The virus titers in culture supernatants were high, and typical coronavirus particles were detected by electron microscopy. Next-generation sequencing of case Wk-521 detected the nearly full-length genome sequence of SARS-CoV-2 with high homology. However, the data also showed contaminated mycoplasma sequences from VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells are superior to other cell lines tested for SARS-CoV-2 isolation. The viral RNA copies in the culture supernatants of VeroE6/TMPRSS2 cells were >100 times greater than those from VeroE6 cells. The results suggest that TMPRSS2 may also play an important role in SARS-CoV-2 cell entry, similar to its role in SARS-CoV. TMPRSS2-expressing cell lines are highly susceptible to SARS-CoV, MERS-CoV, and SARS-CoV-2, making the VeroE6/TMPRSS2 cell line a suitable contributor to the global surveillance of high-risk CoVs. VeroE6/TMPRSS2 cells are easily maintained, suitable for large-scale propagation, and now available from the Japanese Collection of Research Bioresources (JCRB) Cell Bank in Japan. Treatment for mycoplasma is now ongoing in the JCRB Cell Bank.A novel coronavirus, SARS-CoV-2, caused a large respiratory outbreak in Wuhan, China in December 2019. This study shows that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating the virus. The results indicate that TMPRSS2 plays an important role in SARS-CoV-2 infection, similar to its role in SARS-CoV and MERS-CoV. Seven clinical specimens from SARS-CoV-2 infection cases were inoculated into VeroE6/TMPRSS2 cells, which were monitored for cytopathic effect (CPE). In five cases, clear CPE with cell detachment and syncytium formation was observed. The virus titers in culture supernatants were high, and typical coronavirus particles were detected by electron microscopy. Next-generation sequencing of case Wk-521 detected the nearly full-length genome sequence of SARS-CoV-2 with high homology. However, the data also showed contaminated mycoplasma sequences from VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells are superior to other cell lines tested for SARS-CoV-2 isolation. The viral RNA copies in the culture supernatants of VeroE6/TMPRSS2 cells were >100 times greater than those from VeroE6 cells. The results suggest that TMPRSS2 may also play an important role in SARS-CoV-2 cell entry, similar to its role in SARS-CoV. TMPRSS2-expressing cell lines are highly susceptible to SARS-CoV, MERS-CoV, and SARS-CoV-2, making the VeroE6/TMPRSS2 cell line a suitable contributor to the global surveillance of high-risk CoVs. VeroE6/TMPRSS2 cells are easily maintained, suitable for large-scale propagation, and now available from the Japanese Collection of Research Bioresources (JCRB) Cell Bank in Japan. Treatment for mycoplasma is now ongoing in the JCRB Cell Bank.