Lolitrem B is the most potent indole-diterpene mycotoxin produced by Epichloë festucae var. lolii (termed LpTG-1), with severe intoxication cases reported in livestock. To date, there are no in vivo metabolism studies conducted for the mycotoxin. A mouse model assay established for assessing toxicity of indole-diterpenes was used to investigate metabolic products of lolitrem B. Mice were administered lolitrem B at 0.5 and 2.0 mg/kg body weight (b.wt) intraperitoneally before body and brain tissues were collected at 6 h and 24 h post-treatment. Samples were cryoground and subjected to a biphasic or monophasic extraction. The aqueous and lipophilic phases were analysed using liquid chromatography high-resolution mass spectrometry (LC–HRMS); data analysis was performed with Compound Discoverer™ software. A total of 10 novel phase I metabolic products were identified in the lipophilic phase and their distribution in the liver, kidney and various brain regions are described. The biotransformation products of lolitrem B were found to be present in low levels in the brain. Based on structure–activity postulations, six of these may contribute towards the protracted tremors exhibited by lolitrem B-exposed animals.