Calreticulin (CRT) has been described as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However whether CRT directly recognizes aglycone (protein portion) of glycoprotein remains controversial. Our previous study demonstrated that CRT competitively inhibited glucosidase II activity against a hydrophobic substrate not but that against a hydrophilic substrate, implying that CRT recognizes not only glycan structure but also aglycone hydrophobicity. Here to investigate the possibility we prepared ligands for CRT: G1M9-derivatives with different hydrophobicity on the aglycone and gradually denatured immunoglobulin Y (IgY), which harbors G1M9 glycan, and analyzed the interaction of a recombinant CRT with the ligands using thermal shift assay. These results demonstrate that CRT strongly binds to more hydrophobic G1M9-derivative and more denatured IgY, clarifying that CRT more strongly binds misfolded glycoprotein, and reversely releases folded glycoprotein by distinguishing the folding status of glycoprotein in the ER glycoprotein quality control system.