To compare the effects of different carbon sources on physiological aspects, especially medicinal alkaloid biosynthesis and related gene expression in Catharantus roseus (L.) G.Don, we employed sucrose and sorbitol with two concentrations (87.64 mM, the equimolar concentration of sucrose in MS basal medium, and 150 mM) on the plant's shoots in vitro in presence of 100 μM methyl jasmonate. The production of plant alkaloids including vincristine, vinblastine, ajmalicine, vindoline and catharantine and their biosynthetic and regulatory gene expression was measured. Both treatments had incremental effects on alkaloid production, upregulated the mitogen-activated protein kinase3 (MAPK3) and a downstream responsive transcription factor, ORCA3, which resulted in elevated transcript contents of the important genes in terpenoid indol alkaloids biosynthetic pathway including peroxidase1 (PRX1), geissoschizine synthase (GS), strictosidine synthase (STR) and deacetylvindoline acetyltransferase (DAT). Defensive responses such as antioxidant enzymes (catalase, peroxidase and superoxide dismutase) activities and non-enzymatic metabolites (total phenolics, flavonoids and carotenoids) contents increased under both treatments but the effects of sorbitol were stronger. Reduced fresh weight and chlorophylls contents, increased malondialdehyde (MDA) and carotenoid contents were shown after a week under all employed treatments. It seems that replacement of sucrose with sorbitol and also, increased concentrations of both carbon sources via increasing osmotic pressure make stressful conditions for the plant especially in longer times.