The intensifying global warming may increase the impact of heat stress on the dairy industry. Our previous study showed that chromium yeast (CY) alleviated the negative effects of heat stress and improved the lactation performance by increasing milk protein content and yield in mid-lactation dairy cows. This study further investigated whether the increased milk protein after CY supplementation results from the promotion of microbial crude protein (MCP) synthesis by regulating rumen microorganisms and amino acid metabolites. Twelve heat-stressed dairy cows were divided into two treatment groups: one with CY supplementation (0.36 mg Cr/kg DM) and the other without CY supplementation. Samples were collected after eight weeks of formal experiment in a hot summer with the mean temperature-humidity index of 79.0 ± 3.13. Dietary CY supplementation did not affect rumen pH, total volatile fatty acid, acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate, but increased ruminal MCP concentration (