Objective: To evaluate the relationship between serum total homocysteine (tHcy) levels and cerebral small vessel disease (cSVD) in a healthy population. Methods: We included consecutive participants who visited our department for health check-ups between 2006 and 2013. We rated white matter hyperintensity (WMH) volumes using both the Fazekas score and semi-automated quantitative methods. We also evaluated lacunes, cerebral microbleeds (CMBs), and enlarged perivascular spaces (EPVSs) which are involved in cSVD. To assess the dose-dependent relationship between tHcy and cSVD parameters, we scored the burdens of each radiological marker of cSVD. Results: A total of 1,578 participants were included (age: 55 ± 8 years, male sex: 57%). In the multivariable analysis, tHcy remained an independent predictor of the WMH volume (B = 0.209; 95% confidence interval [CI] = 0.033 to 0.385, P = 0.020), presence of CMBs (adjusted odds ratio [aOR] = 2.800; 95% CI = 1.104 to 7.105, P = 0.030), and moderate-to-severe EPVSs (aOR = 5.906; 95% CI = 3.523 to 9.901, P < 0.001) after adjusting for confounders. Furthermore, tHcy had positive associations with periventricular Fazekas score (P = 0.001, P for trend < 0.001), subcortical Fazekas score (P = 0.003, P for trend = 0.005), and moderate-to-severe EPVS lesion burden (P < 0.001, P for trend < 0.001) in a dose-dependent manner. Conclusions: Serum tHcy level is correlated with cSVD development in a dose-dependent manner. These findings provide us with clues for further studies of the pathophysiology of cSVD.