Sterilization of rodent feed is recommended to eliminate potential murine pathogens and minimize microbial variability between batches. Most research institutions sterilize feed using steam/pressure (autoclave) or irradiation. Both methods have advantages and disadvantages that contribute to their suitability, including cost, maintenance, availability, and alterations to the exposed product. Dry heat sterilization, which has been in use for over 75 y, uses higher temperatures and longer sterilization times than steam autoclave and is most often used for delicate instruments or products that would be damaged by water such as powders or oil-based liquids. Dry heat sterilization in vivaria has been limited to date but is gaining popularity due to lower initial purchase and ongoing operational costs as compared with steam autoclaves. Little published information exists on the effects of dry heat sterilization on animal feed. We evaluated the sterility and chemical alterations of a natural ingredient, pelleted, rodent diet (NIH-31) after exposure to dry heat. Feed sterility was achieved using a dry heat exposure temperature of 160 °C (320 °F) for 4 h. This exposure resulted in a significant loss of heat-labile vitamins and significantly more acrylamide production as compared with the nonsterile, irradiated, and autoclaved feed.