Determination of the mechanisms of interspecies transmission is of great significance for the prevention of epidemic diseases caused by emerging coronaviruses (CoVs). Recently, porcine deltacoronavirus (PDCoV) was shown to exhibit broad host cell range mediated by surface expression of aminopeptidase N (APN), and humans have been reported to be at risk of PDCoV infection. In the present study, we first demonstrated overexpression of APN orthologues from various species, including mice and felines, in the APN-deficient swine small intestine epithelial cells permitted PDCoV infection, confirming that APN broadly facilitates PDCoV cellular entry and perhaps subsequent interspecies transmission. PDCoV was able to limitedly infect mice , distributing mainly in enteric and lymphoid tissues, suggesting that mice may serve as a susceptible reservoir of PDCoV. Furthermore, elements (two glycosylation sites and four aromatic amino acids) on the surface of domain B (S1 ) of the PDCoV spike glycoprotein S1 subunit were identified to be critical for cellular surface binding of APN orthologues. However, both domain A (S1 ) and domain B (S1 ) were able to elicit potent neutralizing antibodies against PDCoV infection. The antibodies against S1 inhibited the hemagglutination activity of PDCoV using erythrocytes from various species, which might account for the neutralizing capacity of S1 antibodies partially through a blockage of sialic acid binding. The study reveals the tremendous potential of PDCoV for interspecies transmission and the role of two major PDCoV S1 domains in receptor binding and neutralization, providing a theoretical basis for development of intervention strategies. Coronaviruses exhibit a tendency for recombination and mutation, which enables them to quickly adapt to various novel hosts. Previously, orthologues of aminopeptidase N (APN) from mammalian and avian species were found to be associated with porcine deltacoronavirus (PDCoV) cellular entry . Here, we provide evidence that mice are susceptible to PDCoV limited infection. We also show that two major domains (S1 and S1 ) of the PDCoV spike glycoprotein involved in APN receptor binding can elicit neutralizing antibodies, identifying two glycosylation sites and four aromatic amino acids on the surface of the S1 domain critical for APN binding and demonstrating that the neutralization activity of S1 antibodies is partially attributed to blockage of sugar binding activity. Our findings further implicate PDCoV's great potential for interspecies transmission, and the data of receptor binding and neutralization may provide a basis for development of future intervention strategies.