Background: Our blood bank is a regional blood transfusion centre, which accepts blood only from voluntary donors. Aim: The aim is to study iron status of regular voluntary donors who donated their blood at least twice in a year. Materials and Methods: Prior to blood donation, blood samples of 220 male and 30 female voluntary donors were collected. Control included 100 each male and female healthy individuals in the 18- to 60-year age group, who never donated blood and did not have any chronic infection. In the study and control groups, about 10% subjects consumed non-vegetarian diet. After investigation, 85 males and 56 females having haemoglobin (Hb) levels above 12.5 g/dl were selected as controls. Donors were divided into ≤10, 11-20, 21-50 and >50 blood donation categories. Majority of the donors in >50 donation category donated blood four times in a year, whereas the remaining donors donated two to three times per year. Haematological parameters were measured on fully automatic haematology analyzer, serum iron and total iron-binding capacity (TIBC) by biochemical methods, ferritin using ELISA kits and transferrin using immunoturbidometry kits. Iron/TIBC ratio × 100 gave percentage of transferrin saturation value. Statistical Analysis: Statistical evaluation was done by mean, standard deviation, pair t-test, χ2 and anova (F-test). Results: Preliminary analysis revealed that there was no significant difference in the iron profile of vegetarian and non-vegetarian subjects or controls and the donors donating <20 times. Significant increase or decrease was observed in mean values of various haematological and iron parameters in donors who donated blood for >20 times (P < 0.001), compared to controls. Anaemia, iron deficiency and depletion of iron stores were more prevalent in female donors (P < 0.05) compared to males and especially in those male donors who donated their blood for more than 20 times. Conclusion: Regular voluntary blood donors should receive iron supplementation to prevent iron deficiency and depletion in iron stores.