The Vero cell line is the most used continuous cell line for viral vaccine manufacturing with more than 40 years of accumulated experience in the vaccine industry. Additionally, the Vero cell line has shown a high affinity for infection by MERS-CoV, SARS-CoV, and recently SARS-CoV-2, emerging as an important discovery and screening tool to support the global research and development efforts in this COVID-19 pandemic. However, the lack of a reference genome for the Vero cell line has limited our understanding of host–virus interactions underlying such affinity of the Vero cell towards key emerging pathogens, and more importantly our ability to redesign high-yield vaccine production processes using Vero genome editing. In this paper, we present an annotated highly contiguous 2.9 Gb assembly of the Vero cell genome. In addition, several viral genome insertions, including Adeno-associated virus serotypes 3, 4, 7, and 8, have been identified, giving valuable insights into quality control considerations for cell-based vaccine production systems. Variant calling revealed that, in addition to interferon, chemokines, and caspases-related genes lost their functions. Surprisingly, the ACE2 gene, which was previously identified as the host cell entry receptor for SARS-CoV and SARS-CoV-2, also lost function in the Vero genome due to structural variations.