A comprehensive proteomic study utilizing 2D-DIGE and MALDI-TOF was used to assess the effect of inhibiting two different regulatory mechanisms of telomerase in glioma. RNAi was used to target hTERT and hsp90α. Inhibition of telomerase activity resulted in downregulation of various cytoskeletal proteins with correlative evidence of the involvement of telomerase in regulating the expression of vimentin. Inhibition of telomerase via sihTERT resulted in the downregulation of vimentin expression in glioma cell lines in a grade-specific manner. This study identified novel downstream role of telomerase in regulating the expression of vimentin, thereby affecting tumor progression and metastasis.