Human ACE2 (hACE2) is the key cell attachment and entry receptor for SARS-CoV-2, with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe the emergence of a SARS-CoV-2 strain capable of ACE2-independent infection, and evolution of mouse-adapted SARS-CoV-2 by in vitro serial passaging of virus in co-cultures of cell lines expressing hACE2 and mACE2. Mouse-adapted viruses evolved with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. Mouse-adapted viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates, and caused characteristic lung histopathology. One mouse-adapted virus also evolved to replicate efficiently in several ACE2-negative cell lines across several species, including CRISPR/Cas9 ACE2 knockout cells. An E484D substitution is likely involved in ACE2-independent entry and has appeared in only ≈0.003% of human isolates globally, suggesting it provided no significant selection advantage in humans. ACE2-independent entry reveals a SARS-CoV-2 infection mechanism that has potential implications for disease pathogenesis, evolution, tropism and perhaps also intervention development.