Telomerase is essential for maintaining telomere length and chromosome stability in stem cells, germline cells, and cancer cells. The telomerase ribonucleoprotein complex consists of two essential components, a catalytic protein component and an RNA molecule that provides the template for telomeric repeat synthesis. A pseudoknot structure in the human telomerase RNA is conserved in all vertebrates and is essential for telomerase activity. It has been proposed that this highly conserved structure functions as a dynamic structure with conformational interchange between the pseudoknot and a hairpin with intraloop base pairings. To examine the structural and functional requirements of the pseudoknot structure, we made mutations in the proposed base-paired regions in the pseudoknot. Although mutations that disrupted the pseudoknot P3 helix abolished activity as predicted, mutations that disrupted the intraloop hairpin base pairings did not reduce telomerase activity, indicating that the intraloop hairpin is not required for telomerase function. This functional study thus provides evidence against the previous proposed molecular-switch model of telomerase pseudoknot function and supports a static pseudoknot structure. The mutational analysis further suggests that telomerase RNA can function independent of the proposed intermolecular pairings between pseudoknot regions on two RNA molecules.