Our aim in this study was to measure serum levels of methylarginines and related metabolites in patients with ankylosing spondylitis (AS), moreover, to investigate the relationship between these parameters and various clinical and laboratory parameters of patients with AS. The study included 60 patients with AS and 60 healthy volunteers. Serum asymmetric dimethylarginine (ADMA), L–N monomethylarginine (L-NMMA), symmetric dimethylarginine (SDMA), arginine (Arg), homoarginine (hArg), ornithine, and citrulline concentrations were measured with tandem mass spectrometry. In addition, participants were divided into three groups according to the treatment regimen: TNF-α inhibitor group (n = 25), conventional therapy group (n = 35), and control group (n = 60). These groups were compared in terms of serum levels of methylarginine pathway metabolites and various biochemical parameters. It was found that total methylated arginine load significantly increased in patients with AS (p < 0.001), and the Arg/ADMA ratio was positively correlated with HDL levels and negatively correlated with glucose, ESR, total cholesterol, triglyceride, and LDL levels. In addition, serum ADMA, SDMA, total methylated arginine load, and CRP levels were lower (p < 0.05) in the TNF-α group compared to the conventional treatment group. To the best of our knowledge, this is the first study to comprehensively investigate serum methylarginine levels in patients with AS. Elevated total methylated arginine load and decreased global arginine bioavailability ratio (GABR) indicate that NO metabolism is impaired in patients with AS. Therefore, the increased cardiovascular risk in patients with AS may be related to the decreased NO production or bioavailability due to the elevated total methylarginine load.