Bats are a major reservoir of emerging infectious diseases and harbour deadly viruses such as Ebola, SARS, MERS and Hendra virus which pose serious threats to animal and human health. We hypothesise that the bat adaptive immune system contributes to their ability to co-exist asymptomatically with these viruses. One of the key adaptive immune responses is the presentation of antigens by major histocompatibility complex (MHC) class I molecules to cytotoxic CD8⁺ T cells. Using a proteomic approach, we have characterised the proteins and peptides bound to three distinct bat MHC class I molecules. We determined that these bat MHC class I molecules, like their human and mouse homologues, associate with a peptide-loading complex to facilitate optimal ligand selection. Using a peptidomics approach, we also characterised the first repertoires of bat MHC class I bound peptides. These peptides ranged in length from 8-15 amino acid residues and motif analysis of the endogenous peptides revealed strong amino acid biases at various anchor positions. Furthermore, we were able to identify Hendra virus-derived peptides from infected bat cells which also display this binding motif, suggesting these motifs may be exploited to predict epitopes for vaccine development and immunological studies. In conclusion, this study provides fundamental insights into the antigen processing and presentation pathways of bats, which ultimately can be used to understand viral control.