Bats are natural reservoirs of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Scotophilus bat CoV-512 demonstrates potential for cross-species transmission because its viral RNA and specific antibodies have been detected in three bat species of Taiwan. Understanding the cell tropism of Scotophilus bat CoV-512 is the first step for studying the mechanism of cross-species transmission. In this study, a lentivirus-based pseudovirus was produced using the spike (S) protein of Scotophilus bat CoV-512 or SARS-CoV as a surface protein to test the interaction between coronaviral S protein and its cell receptor on 11 different cells. Susceptible cells expressed red fluorescence protein (RFP) after the entry of RFP-bound green fluorescence protein (GFP)-fused S protein of Scotophilus bat CoV-512 (RFP-Sco-S-eGFP) or RFP-SARS-S pseudovirus, and firefly luciferase (FLuc) activity expressed by cells infected with FLuc-Sco-S-eGFP or FLuc-SARS-S pseudovirus was quantified. Scotophilus bat CoV-512 pseudovirus had significantly higher entry efficiencies in Madin Darby dog kidney epithelial cells (MDCK), black flying fox brain cells (Pabr), and rat small intestine epithelial cells (IEC-6). SARS-CoV pseudovirus had significantly higher entry efficiencies in human embryonic kidney epithelial cells (HEK-293T), pig kidney epithelial cells (PK15), and MDCK cells. These findings demonstrated that Scotophilus bat CoV-512 had a broad host range for cross-species transmission like SARS-CoV.Bats are the natural reservoirs of SARS-CoV, MERS-CoV, and HCoV-NL63. SARS-CoV and HCoV-NL63 gain cellular entry through angiotensin-converting enzyme 2 (ACE2) [9], whereas MERS-CoV utilizes dipeptidyl peptidase-4 (DPP4) as its entry receptor [10]. The entry receptor is considered a hallmark of coronaviral cross-species transmissibility. SARS-related CoV isolated from Rhinolophus bats can use the ACE2 of humans, civets, and Rhinolophus bats as its cell receptor to infect cells originated from human and many other animal species [11]. Both MERS-CoV and bat CoV-HKU4 can use bat and human DPP4 to infect cells originating from humans, camels, bats, and other animal species [12,13] and HCoV-NL63 can replicate in the lung cell line from tricolored bats [8].Little information is available regarding cross-species events for animal CoVs. A previous study detected Scotophilus bat CoV-512 in four different bat species along with Miniopterus bat CoV-1A and Rhinolophus SARS-related CoV in Taiwan through the reverse transcription polymerase chain reaction (RT-PCR) [14]. Antibodies specific to the nucleocapsid (N) protein fragments of Scotophilus bat CoV-512 were detected in serum collected from three bat species, namely Scotophilus kuhlii, Miniopterus fuliginosus, and Rhinolophus monoceros [15]. A close relationship and possible gene recombinants between Scotophilus bat CoV-512 and PEDV were observed through sequence alignments [14,16]. The results of the cell entry assay also indicated that PEDV can infe...