Calreticulin (CRT) is a lectin chaperone present in the lumen of the endoplasmic reticulum. It interacts with various glycoproteins by binding via their attached Glc(1)Man(9)GlcNAc(2) moiety. To provide further insight into these lectin-glycan interactions, we are investigating the interaction of CRT with various sugars. We have earlier modeled the complex between CRT and the Glc(1)Man(3) tetrasaccharide, a derivative of the native Glc(1)Man(9)GlcNAc(2) sugar moiety. Here, we have systematically mutated the residues implicated by the model in the interaction of CRT to its sugar substrates and categorized the role played by each of the subsites of calreticulin toward the glycan binding. The CRT mutants Y109F and D135L did not show any binding to the sugar substrates interacting with the wild-type protein, demonstrating the great importance of these residues in the carbohydrate-binding site of CRT. Also, D317L and M131A showed weak affinity toward the trisaccharide. The mutation of residues from the primary binding site of CRT, i.e., those interacting with glucose, appears to be far less tolerated as compared to mutations in residues that interact with the mannose residues of the glycan. Also, methyl-2-deoxy-glucopyranosyl-alpha(1-->3)-mannopyranoside failed to bind, asserting to the significance of the interactions between the primary binding site of CRT and the 2'-OH of the glucose residue of the oligosaccharide substrate in generating specificity for this recognition. These studies provide detailed molecular insight into the sugar binding specificity of CRT.