1-Ethoxy-2,3-difluoro-4-(trans-4-propylcyclohexyl)benzene (EDPrB) is a typical fluorinated liquid-crystal monomer (LCM). LCMs contaminants are becoming increasingly concerning due to their potential persistence, bioaccumulation, toxicity, and broad prevalence in environmental and human samples. However, LCM metabolism is poorly understood. Herein, by introducing selected EDPrB into the appropriate liver microsomes in vitro, we examined the metabolic pathways of LCM in humans, rats, pigs, Cyprinus carpio, crucian carp, and Channa argus. A total of 20 species-dependent metabolites were identified and structurally elucidated by gas and liquid chromatography-high resolution mass spectrometry for the first time. Dealkylation, H-abstraction, and hydroxylation reactions are the primary metabolic pathways. Half of these in vitro metabolites were found in the urine, serum, and fecal samples of Sprague–Dawley rats exposed to EDPrB. Toxicity predictions indicate that 17 metabolites can be classified as toxic. According to the Ecological Structure Activity Relationships (ECOSAR), a number of metabolites exhibit equivalent or greater aquatic toxicity to that of EDPrB. Toxicity Estimation Software Tool (T.E.S.T.) predicts that some metabolites exhibit developmental toxicity and mutagenicity in rats. These findings suggest that biotransformation should be particularly emphasized, and more toxicological and monitoring studies should be performed to assess the ecological and human safety of LCMs.