Heat stress (HS) decreases milk yield and deleteriously alters milk composition in dairy cows. This study examined the ruminal metabolic response of dairy cows to HS using a combination of LC-MS, GC-MS, and ¹H NMR. Four multiparous Holstein dairy cows (101 ± 10 DIM; 574 ± 36 kg of BW, 38 ± 2 kg of milk/d) were randomly assigned to 4 environment chambers with a crossover design. Cows were either subjected to HS [HS: 36°C with light and 32°C without light; THI = 87.2 and 81.8] or kept under thermal neutral conditions [TN: 20°C; THI = 65.5] for 9 d for adaptation and then for another 9 d of pair-feeding to eliminate confounding effects of dissimilar feed intake. There was a 30-d washout period between periods. Rumen fluid was collected at 1000 h (after feeding) on d 9. All data were analyzed using R, SIMCA-P 13.0, and SIEVE software. Metabolites were identified by m/z value matched to the NIST database. A student t-test was used to search for changed metabolic profiles with Variable Importance in the Projection (VIP) greater than 1 in an OPLS-DA model and a P-value less than 0.05. The OPLS-DA results showed that all the metabolic profiles in the rumen fluid changed were separated into 2 groups in response to HS and TN. Based on LC-MS, GC-MS and 1H NMR results, the metabolites of glucose, galactose, glycerol, butyrate, glucosamine, heptacosane and hentriacontane were increased by HS (VIP >1, P < 0.05, Fold change >0), while fatty acids and amino acids were decreased (VIP >1, P < 0.05, Fold change <0). Most of the rumen metabolites affected by HS were related to several metabolic pathways, including urine cycle pathway, metabolism of amino acids, tryptophan metabolism and citrate cycle, which likely affect the precursor supply for milk component synthesis. These findings indicate that the use of multiple metabolomics platforms permits a far more detailed understanding of HS-induced metabolic changes in rumen digestion: potential mechanism by which HS decreases milk production and component synthesis.