Background: Complex metabolic pathways may exist for atherosclerosis. We examined metabolomic signatures that reflect mechanistic pathways associated with carotid atherosclerosis in apparently healthy men. Methods: A cross-sectional study was conducted in 675 Japanese men (46-83 years) from the SESSA Study (2010-2014). A total of 468 serum metabolites were quantified by liquid chromatography mass spectrometry. The mean intima-media thickness (IMT) and total number of plaques from carotid arteries were measured using B-mode ultrasound. All metabolites were standardized prior to the analysis. We used partial least square regression and variable importance projection (VIP) method to extract the principal metabolites most associated with the mean IMT and total number of plaques. We used a multivariable linear model for the log-mean IMT and a multivariable Poisson model for the total number of plaques to assess the association between individual extracted serum metabolites and carotid measures. Each metabolite multivariable model adjusted for age, body mass index, smoking and drinking status, medications for hypertension, diabetes and dyslipidemia, and systolic blood pressure, high- and low-density lipoprotein cholesterols and hemoglobin A1c. We used false discovery rate adjusted p-values to control for multiple testing. Results: The average mean IMT and total number of plaques were 0.80 mm (SD: 0.16) and 2.06 (range: 0-14), respectively. There were 191 metabolites with a VIP value of one or higher for mean IMT. None of these metabolites was significantly associated with the log-mean IMT. On the other hand, there were 209 metabolites with a VIP value of one or higher for the total number of plaques. Among them, SulfoHexCer(d18:2/24:0(OH)) and SulfoHexCer(d18:2/16:0(OH)) had an inverse association with the total number of plaques (Figure 1). Conclusion: We identified distinct metabolites associated with the total number of plaques. The results suggest an important role of sphingolipids in carotid plaque formation.