Choline and trimethylamine N-oxide (TMAO) have been considered as factors associated with cardiovascular disease (CVD). TMAO is produced from trimethylamine (TMA) oxidation by flavin-containing monooxygenase 3. Flavin-containing monooxygenases (FMOs) are enzymes that catalyze oxygenation of a wide range of compounds and drugs. Human and mouse livers, are included other members of FMO family, FMO4 and FMO5. Expression of FMO5 in our liver is approximately equal to FMO3 expression. FMO3 and FMO5 enzymes are known to be involved in lipids and glucose metabolism. In this study, we investigated the effects of choline, TMA and TMAO on FMO3 and FMO5 expression. For this purpose, these compounds were injected to female NMRI mice as intraperitoneally and then liver tissues were collected to evaluate of FMO3 and FMO5 expression. Trimethylamine administration resulted in 8-fold (P<0.0001) increase in FMO3 mRNA levels and an 2.5-fold (P<0.0001) increase in FMO3 protein compared to controls (mean different equal to 6.87 for mRNAs and 1.67 for protein). A significant decrease (approximately 45% (P<0.05)) in FMO3 mRNA was observed following choline administration (mean difference of FMO3 mRNA levels between treatment and control groups equal to 0.576). FMO5 mRNA levels were decreased to 50% than to controls (P=0.003) after TMA treatment. Overall, our data indicate that FMO3 and FMO5 expression is influenced by TMA and choline.