Background: Ribonuclease-L (RNase-L) was known to be a ubiquitous enzyme involved in several cellular functions, especially innate immunity. It was recently shown to participate in adipogenesis in rodents. Here, we developed a method to measure serum levels of RNase-L and analyzed the relationship between RNase-L and metabolic syndrome (MetS). Methods: A total of 396 subjects were recruited from a health check-up program. An in-house RNase-L immunoassay was developed. The serum RNase-L levels of these subjects were measured, and the association of MetS-related factors with RNase-L levels was assessed. Results: The mean serum level of RNase-L of the subjects with MetS were lower than those without (16.5 +/- 6.4 vs. 18.4 +/- 8.0 mu g/ml, P = 0.018). The subjects with central obesity, elevated blood pressure, or impaired fasting glucose also had lower serum RNase-L levels in comparison to those without. In multivariate linear regression analysis, diastolic blood pressure (beta = -0.129, P = 0.024) and high-density lipoprotein cholesterol (HDL-C) (beta = 0.127, P = 0.036) were related to serum RNase-L. For every 5 mu g/ml increase in serum RNase-L levels, it is associated with a reduced risk of MetS (OR 0.83, 95% CI 0.71-0.98, P = 0.028), central obesity (OR 0.82, 95% CI 0.71-0.94, P = 0.005), or low HDL-C (OR 0.86, 95% CI 0.74-1.00, P = 0.042). Moreover, age is inversely related to serum RNase-L levels in various analyses. Conclusions: The serum RNase-L levels were inversely associated with MetS, unfavorable metabolic profiles, and age.