The present study was carried out to explore the potential effects of multi-walled carbon nanotubes (MWCNTs) on callus induction and secondary metabolism in Satureja khuzestanica. Leaf segments were aseptically cultured in B5 basal medium with different MWCNTs concentrations (0, 25, 50, 100, 250 and 500 lgml1). The calli morphogenic responses were measured and the contents of phenolics, flavonoids, rosmarinic acid (RA), caffeic acid (CA), and the activity of polyphenol oxidase (PPO), L-phenylalanine ammonia-lyase (PAL) and peroxidase (POD) were quantified. Moreover, antioxidant activities of calli extract were assayed. Calli growth improved significantly with the increase of MWCNTs concentration, peaked at 50 lgml1, and then followed a rapid decrease at 500 lgml1. However, metabolic effects observed following exposure to MWCNTs particularly at 100 lgml1 tended to be more pronounced than all other treatments, exhibiting significant induction of antioxidant activity with the lowest IC50 value. Maximum oxidative stress index (H2O2) and the highest PPO and POD activities were observed on the media treated with 500 lg ml1 MWCNTs. Our findings suggest for the first time that use of MWCNTs at specific levels could act as a novel elicitor for in vitro biosynthesis of valuable secondary metabolites and antioxidant drugs.