Restless Legs Syndrome (RLS) can be defined as a sleep disorder. However, whether changes in the serum vitamin B12 levels are involved in the pathophysiological mechanism of RLS remains unclear. Our study aimed to determine whether vitamin B12 levels are independently related to the occurrence of RLS. The serum vitamin B12 levels of 80 patients with RLS and 80 age- and gender-matched healthy controls (HC) were retrospectively analyzed. Serum vitamin B12 levels in the RLS group were significantly reduced, while the levels of creatinine, and homocysteine were higher (P<0.05). In addition, multivariate logistic regression revealed serum vitamin B12 to be independently associated with RLS (p<0.05; odds ratio=0.97; 95% confidence interval: 0.96-0.98). Pearson correlation analysis indicated that serum vitamin B12 level was negatively correlated with the International Restless Legs Scales (IRLS) score, and the 24-item Hamilton Depression Rating Scale (HAMD24) score (r=-0.025, P=0.023, r=-0.295, P=0.001). Patients with RLS had significant vitamin B12 deficiency compared to HC. Such deficiency significantly affects severity of symptoms and depression symptoms. In addition, decreased serum vitamin B12 levels are independently associated with the development of RLS, which illustrates the complex relationship between vitamin B12 and RLS. Prospective vitamin B12 treatment studies are needed to confirm this relationship and to evaluate the efficacy of vitamin B12 as a treatment for RLS patients.