Indoor storage of honey bees (Apis mellifera L.) during winter months has been practiced for decades to protect colonies from the adverse effects of long, harsh winter months. Beekeepers have recently employed indoor storage to reduce labor, feeding costs, theft, and woodenware degradation. Despite the growing number of colonies stored indoors, national survey results still reveal high losses. Varroa mites (Varroa destructor Anderson and Trueman) are the most critical threat to colony winter survival and health of colonies because they contribute to the transmission of viruses and colony mortality. To investigate the effect of high CO₂ on varroa mites during the indoor storage of honey bees, 8-frame single deep colonies were stored in two separate environmental chambers at 4°C each. One environmental chamber was set at 8.5% CO₂ (high CO₂), while the other was set at low CO₂ (0.12%). Dead and falling mites were collected and counted from the bottom of individual colonies weekly during the experiment. There was a significant difference in mite mortality of colonies with high CO₂ compared to colonies held at low CO₂. These results indicated that high CO₂ could increase mite mortality during the period of indoor storage, potentially improving honey bee health coming out of the winter months. Our research offers a critical addition to beekeepers' tools for managing varroa mite populations.