Chronic exposure to high inorganic As levels in drinking water has been related to many diseases, including type 2 diabetes mellitus (T2D). The association with low and moderate As levels, however, remains controversial and has yet not been studied in European populations. This study aimed to investigate possible association between As exposure and biomarkers of T2D in Croatian population. Observation recruited 86 adults from Eastern Croatia, where groundwater is contaminated with inorganic As, and 116 adults from Western Croatia, where As levels in drinking water are low. Both populations were divided in patient groups (T2D or prediabetes) and healthy controls. Exposure was assessed by determining total As in blood and urine and As metabolites in urine. Eastern Croatian population had a significantly higher content of As in urine than Western, whereas the opposite was true for arsenobetain. Total As and As metabolites in urine positively correlated with hemoglobin A1c (HbA1c) and negatively with albuminuria. This study provides important preliminary data on the levels of As in urine and blood and their association with biomarkers of T2D in Croatian population exposed to low or moderate levels of As through drinking water as a solid basis for further research of the pathophysiological effects of such As exposure on the status and complications of diabetes. Display omitted •This is first study providing data on urinary levels of As species in Croatian population.•Correlation between As in urine and blood with biomarkers of diabetes were investigated.•Higher concentrations of As species in urine were associated with geographical origin.•As levels in urine positively correlated with HbA1c and negatively with albuminuria