An efficient method of regeneration for antidiabetic plant (Stevia rebaudiana) has been established for healthy biomass and main steviol glycosides (SGs) production, using different PGRs and agar concentrations. Higher callus induction (93.3%) was recorded when leaf explants were placed on an MS medium supplemented with 3.5 gL(-1) agar and 2.0 mgL(-1) 2,4-D. The addition of 7.0 gL(-1) agar and BA (1.0, 2.0 and 4.0 mgL(-1)) significantly (P<0.01) influences shooting response (100%). A maximum mean shoot length (13.03 cm) and 28 shoots per explant were observed on a medium containing 1.0 mgL(-1) BA. However, the maximum number of leaves (132.67) was encouraged by the addition of BA (1.0 mgL(-1)) and Kin (1.0 mgL(-1)). Lower agar (3.5 gL(-1)), IAA (2.0 mgL(-1)), and NAA (2.0 mgL(-1)) concentrations significantly influence the rooting percent (100%), the mean root length (2.9 cm), and the number of roots per plantlet (26.3). These plantlets were successfully acclimatized in the soil. The BA (3.0 mgL(-1)) in combination with Kin (3.0 mgL(-1)) and 3.5 gL(-1) agar increases dulcoside-A content (Dul-A; 71.8 μg/g-DW) in shoots compared to control (50.81 μg/g-DW). Similar PGRs with 7.0 gL(-1) significantly increases the production of steviosides (Stev. 82.48 μg/g-DW). A higher rebaudioside-A content (Reb-A; 12.35 μg/g-DW) was observed in shoots that underwent the addition of BA (1.0 mgL(-1)) and 7.0 gL(-1) agar than in control (07.39 μg/g-DW). Hereby, we developed an efficient and cost-effective method for regeneration and major SGs production, which could be helpful for future studies on this species. Copyright © 2013. Published by Elsevier SAS.