STUDY QUESTION: Are levels of trimethylamine-N-oxide (TMAO) in human follicular fluid (FF) related to IVF outcomes? SUMMARY ANSWER: Higher levels of TMAO are a negative predictor of oocyte fertilization and embryo quality. WHAT IS KNOWN ALREADY: TMAO is a metabolic product of dietary choline and l-carnitine produced via subsequent enzymatic modifications by the intestinal microbiota and hepatocytes. TMAO promotes inflammatory and oxidative stress pathways and has been characterized as a causative biomarker for the development of cardiometabolic disease. STUDY DESIGN, SIZE, DURATION: For the present cross-sectional study, samples (FF and plasma) from 431 modified natural cycle (MNC)-IVF cycles of 132 patients were collected prospectively between October 2014 and March 2018 in a single academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS: TMAO and its precursors (choline, l-carnitine and gamma-butyrobetaine) were measured by ultra-high-performance liquid chromatography/mass spectrometry in (i) matched FF and plasma from 63 MNC-IVF cycles, in order to compare metabolite levels in the two matrices and (ii) FF from 232 MNC-IVF cycles in which only one oocyte was retrieved at follicular puncture. The association between metabolite levels and oocyte fertilization, embryo fragmentation percentage, embryo quality and the occurrence of pregnancy was analyzed using multilevel generalized estimating equations with adjustment for patient and cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: The level of choline was higher in FF as compared to matched plasma (P < 0.001). Conversely, the levels of TMAO and gamma-butyrobetaine were lower in FF as compared to plasma (P = 0.001 and P = 0.075, respectively). For all metabolites, there was a positive correlation between FF and plasma levels. Finally, levels of TMAO and its gut-derived precursor gamma-butyrobetaine were lower in FF from oocytes that underwent normal fertilization (TMAO: Odds ratio [OR] 0.66 [0.49-0.90], P = 0.008 per 1.0-μmol/L increase; gamma-butyrobetaine: OR 0.77 [0.60-1.00], P = 0.047 per 0.1-μmol/L increase) and developed into top-quality embryos (TMAO: OR 0.56 [0.42-0.76], P < 0.001 per 1.0-μmol/L increase; gamma-butyrobetaine: OR 0.79 [0.62-1.00], P = 0.050 per 0.1-μmol/L increase) than in FF from oocytes of suboptimal development. LIMITATIONS, REASONS FOR CAUTION: The individual contributions of diet, gut bacteria and liver to the metabolite pools have not been quantified in this analysis. WIDER IMPLICATIONS OF THE FINDINGS: More research on the contribution of diet and the effect of gut bacteria on FF TMAO is warranted. Since TMAO integrates diet, microbiota and genetic setup of the person, our results indicate potential important clinical implications for its use as biomarker for lifestyle interventions to improve fertility. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this project. The Department of Obstetrics and Gynecology of the University Medical Center Groningen received an unrestricted educational grant of Ferring Pharmaceutical BV, the Netherlands. The authors have no other conflicts of interest. TRIAL REGISTRATION NUMBER: Netherlands Trial Register number NTR4409.