<p>Levodopa (LD) remains the cornerstone of treatment for Parkinson’s disease (PD), but chronic LD therapy has been associated with elevated plasma homocysteine levels. Homocysteine reflects alterations in amino acid and one-carbon metabolism. Catechol-O-methyltransferase inhibitors (COMT-Is) reduce peripheral LD methylation; however, their association with homocysteine levels across different LD doses has not been fully characterized. In this prospective cross-sectional study, 262 patients with PD receiving oral LD/dopa decarboxylase inhibitor therapy were enrolled at two centers in Japan. Patients were stratified by COMT-I use (opicapone or entacapone). Plasma homocysteine and serum vitamin B6, vitamin B12, and folate levels were measured. Multivariable linear regression using log-transformed homocysteine as the outcome variable was performed, adjusting for demographic, clinical, pharmacological, and biochemical factors. Dose-stratified rolling range analyses examined LD dose–dependent associations. COMT-I use was independently associated with lower plasma homocysteine levels (β = −0.166, p = 0.00024). Higher vitamin B12 and folate levels were also inversely associated with homocysteine. In dose-stratified analyses, the homocysteine-lowering effect of COMT inhibition became apparent at LD doses above approximately 450 mg/day, whereas no significant differences were observed at lower doses. COMT inhibitor use is independently associated with lower plasma homocysteine levels in patients with PD receiving LD therapy, particularly at higher LD doses, supporting a metabolic role of COMT-mediated LD methylation.</p>