Abstract: Stevia rebaudiana is a well known natural sweetener. The sweetness is due to steviol glycosides, the glucosylated steviol derivatives of steviol glycoside biosynthesis pathway. In tissue culture conditions, sucrose is utilized as carbon source. In present study, we analyzed the effect of varying sucrose (1, 3 and 5%) concentrations on the genes involved in steviol glycoside biosynthesis pathway and content of steviol glycosides. The higher endogenous sucrose content in 5% sucrose treated plants than that in 3 or 1% treated plants suggested the uptake of exogenously available sucrose by plants. The transcript expression profiling of genes involved in steviol glycoside biosynthesis pathway showed an overall increase in the expression of pathway specific genes; CDPS, KS, KO, UGT85C2 and UGT76G1 in 5% sucrose treated plants compared to that in 1 and 3% treated plants. Furthermore, the quantitative estimation of steviol glycosides in leaves revealed that approximately 4.5 times higher glycosides accumulated in 5% sucrose treated plants than that in 3 and 1% treated plants. However, 1 and 5% sucrose was to be affecting stomatal and trichome density, including the germination rate in comparison to 3% sucrose. Present work thus suggests that sucrose might be acting as an enhancer of transcriptional trigger to the genes of steviol glycoside biosynthesis pathway that could positively manipulate the production of steviol glycosides.