Manganese (Mn) is an essential dietary nutrient for human health serving as a cofactor for many enzymes; however, exposure to excessive quantities of Mn may lead to toxicity with symptoms analogous to Parkinson's disease (PD). Population-based biomonitoring is an effective tool for characterizing the body burden of environmental or occupational pollutants, including Mn. The objective of this cross-sectional study was to (1) estimate reference values (RV) for blood Mn in an adult population and (2) assess the variables that were associated with higher blood metal levels. A total of 947 adults, aged 40 years or older, were randomly selected in a city in Southern Brazil. Information on socioeconomic, dietary, lifestyle, and occupational background was collected by trained interviewers. Blood Mn levels (μg/L) were analyzed by inductively coupled plasma-mass spectrometer (ICP-MS). The RV for blood Mn concentrations were obtained from the upper limits of the 95% confidence intervals (CI) of the 95th percentile distributions. Cluster analysis was performed to identify variables associated with high or low blood Mn concentrations. The RV for blood Mn levels in this study were 18.54 and 20.15 μg/L for men and women, respectively. Mn blood concentrations decreased with age and were higher in females compared to males. No marked association was noted between blood Mn and smoking or drinking habits, education levels and socioeconomic status. Diastolic blood pressure was higher in a group of women approximately 54 years of age associated with elevated blood Mn levels. Important reference data stratified by demographic and lifestyle factors that may prove useful for future surveillance of environmental exposure to Mn and health risks associated with this metal are presented.