Objective DUF538 (domain of unknown function 538) domain containing proteins are known as putative hypothetical proteins in plants. Until yet, there is no much information regarding their structure and function.Methods In the present research work, the homologous structures and binding potentials were identified between plant/mammalian lipocalins and plant DUF538 protein by using bioinformatics and experimental tools including molecular dynamics simulation, molecular docking and recombinant technology-based techniques.Results Molecular docking analysis of their interactions with lipidic ligands including cholesterol and palmitic acid revealed the similar and comparable binding potentials between DUF538 and lipocalin proteins. Both the test proteins were found to have more affinity to cholesterol molecule in compare to palmitic acid. By using recombinant technology-based experiments, the heterologously expressed and purified fused product of DUF538 protein exhibited about 61% cholesterol binding ability.Conclusion As a conclusion, plants DUF538 protein family was predicted to be the structural and may be the functional homologues of plants/animals lipocalin superfamily.