OBJECTIVE: Long-term intake of proton pump inhibitors (PPIs) might increase the risk of cardiovascular events. One suggested mechanism is that PPIs inhibit the enzyme dimethylarginine dimethylaminohydrolase (DDAH), and thereby block the degradation of endothelial asymmetrical dimethylarginine (ADMA). Excess ADMA in turn leads to impaired endothelial nitric oxide (NO) generation. So far, this mechanism has only been established in human cell cultures. This study examined that pathway in a human population. Previous studies that examined this pathway in human populations measured circulating ADMA, and found no association with PPI use and excess plasma ADMA. But in a recent study plasma ADMA was not correlated with intracellular ADMA. We therefore focused on changes in plasma citrulline as an indicator for potential DDAH inhibition. METHODS: We analyzed the association between regular daily PPI intake and flow-mediated dilation (FMD) of the brachial artery as well as plasma concentrations of citrulline, arginine, ADMA, and symmetric dimethylarginine using inverse probability weighting to adjust for confounding and censoring. Data that had been collected between 2008 and 2012 of 1,298 participants from two independent cohorts of the population-based Study of Health in Pomerania was used. RESULTS: 87 participants (57.5% female) were regular daily users of PPIs. In the fully adjusted models, associations were identified for FMD and plasma citrulline concentrations. PPI users revealed a 0.99% (95% CI: -1.96 to -0.02) lower FMD and 3.03 µmol/l (95% CI: -4.96 to -1.10) lower plasma citrulline levels as compared to non-users. CONCLUSION: Our data provide evidence that long-term intake of PPIs might inhibit human DDAH activity, resulting in impaired endothelial NO production and reduced vascular function. In the long run this might explain an increased risk for cardiovascular diseases associated with long-term PPI use.