Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) causes a severe respiratory disease in humans. The MERS-CoV spike (S) glycoprotein mediates viral entry into target cells. For this, MERS-CoV S engages the host cell protein dipeptidyl peptidase 4 (DPP4, CD26) and the interface between MERS-CoV S and DPP4 has been resolved on the atomic level. Here, we asked whether naturally-occurring polymorphisms in DPP4, that alter amino acid residues required for MERS-CoV S binding, influence cellular entry of MERS-CoV. By screening of public databases, we identified fourteen such polymorphisms. Introduction of the respective mutations into DPP4 revealed that all except one (Δ346-348) were compatible with robust DPP4 expression. Four polymorphisms (K267E, K267N, A291P and Δ346-348) strongly reduced binding of MERS-CoV S to DPP4 and S protein-driven host cell entry, as determined using soluble S protein and S protein bearing rhabdoviral vectors, respectively. Two polymorphisms (K267E and A291P) were analyzed in the context of authentic MERS-CoV and were found to attenuate viral replication. Collectively, we identified naturally-occurring polymorphisms in DPP4 that negatively impact cellular entry of MERS-CoV and might thus modulate MERS development in infected patients. © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd.