The gene therapy field has advanced in recent years with five recombinant adeno-associated virus (rAAV) based products winning Food and Drug Administration (FDA) approval. As the number of therapeutic applications and overall production demands for rAAV increase, it is valuable to evaluate rAAV production in different production cells. Chinese hamster ovary (CHO) cells have been a robust host for biomolecule manufacturing for more than 35 years. However, there is no report to our knowledge describing the use of CHO cells for rAAV production. In this study, we examined the ability of CHO cells to produce rAAV using a transient plasmid transfection approach. Our results demonstrated that CHO is capable of producing rAAV with detectable viral fundamental components including viral RNAs, proteins, and rAAV viral particles. We identified the expression of cap proteins as one of the limiting factors for rAAV production in CHO cells. We therefore added an additional cytomegalovirus (CMV)-Cap plasmid to the CHO transfection. After increasing cap protein expression, we detected rAAV titers as high as 3 × 10 viral genomes for every 2 × 10 capsids in CHO cells using a quintuple transfection method (standard AAV2 Rep/Cap, helper, gene of interest plasmids, plus CMV-E1, and CMV-Cap plasmids) with comparable full particle percent (average 15%) to that of human embryo kidney (HEK)-derived rAAV. Our study provides a foundation for potential rAAV production in CHO cells.