Hyperthermia induced by 3,4-methylenedioxymethamphetamine (MDMA) can be life-threatening. Here, we investigate the role of the gut microbiome and TGR5 bile acid receptors in MDMA-mediated hyperthermia. Fourteen days prior to treatment with MDMA, male Sprague-Dawley rats were provided water or water treated with antibiotics. Animals that had received antibiotics displayed a reduction in gut bacteria and an attenuated hyperthermic response to MDMA. MDMA treated animals showed increased uncoupling protein 1 (UCP1) and TGR5 expression levels in brown adipose tissue and skeletal muscle while increased expression of UCP3 was observed only in skeletal muscle. Antibiotics prior to MDMA administration significantly blunted these increases in gene expression. Furthermore, inhibition of the TGR5 receptor with triamterene or of deiodinase II downstream of the TGR5 receptor with iopanoic acid also resulted in the attenuation of MDMA-induced hyperthermia. MDMA-treatment enriched the relative proportion of a Proteus mirabilis strain in the ceca of animals not pre-treated with antibiotics. These findings suggest a contributing role for the gut microbiota in MDMA-mediated hyperthermia and that MDMA treatment can trigger a rapid remodeling of the composition of the gut microbiome.