Calreticulin (CRT) is a soluble, lectin chaperone found in the endoplasmic reticulum of eukaryotes. It binds the N-glycosylated polypeptides via the glycan intermediate Glc(1)Man(5-9)GlcNAc(2), present on the target glycoproteins. Earlier we have studied interactions of substrate with CRT by isothermal titration calorimetry (ITC) and molecular modeling, to establish that CRT recognizes the Glcalpha1-3 linkage and forms contacts with each saccharide moiety of the oligosaccharide Glcalpha1-3Manalpha1-2Manalpha1-2Man. We also delineated the amino acid residues in the sugar binding pocket of CRT that play a crucial role in sugar-CRT binding. Here, we have used mono-deoxy analogues of the trisaccharide unit Glcalpha1-3Manalpha1-2Man to determine the role of various hydroxyl groups of the sugar substrate in sugar-CRT interactions. Using the thermodynamic data obtained by ITC with these analogues we demonstrate that the 3-OH group of Glc1 plays an important role in sugar-CRT binding, whereas the 6-OH group does not. Also, the 4-OH, 6-OH of Man2 and 3-OH, 4-OH of Man3 in the trisaccharide are involved in binding, of which 6-OH of Man2 and 4-OH of Man3 have a more significant role to play. This study sheds light further on the interactions between the substrate sugar of glycoproteins and the lectin chaperone CRT.