Oxidative stress induced changes in conformational properties of proteins are potential causes of functional impairment in protein system. This phenomenon becomes an important study area when the affected proteins present a role in disease aetiology. The current study was aimed at studying seminal plasma protein conformation changes under higher Reactive oxygen species levels. A total of 100 semen samples (control: n = 50 and infertile n = 50) were used after routine semen parameter analysis. Samples were analysed for their total protein content, ROS levels, total antioxidant capacity and polyacrylamide gel electrophoresis was performed under native conditions. A protein band of molecular weight less than 14 KDa was seen differentially in infertile sample presented with higher ROS levels(>72.6 μg formazan/107 sperm cells). MALDI analysis identified the protein to be Tumor Necrosis Factor alpha super family 18 (TNFα-18) ligand. Further analysis of biophysical properties of the protein showed a significant increase in surface hydrophobicity. Binding of seminal plasma TNFα-18 with ANS dye resulted in an increased fluorescence and broadening of emission curves in infertile samples. Changes in structural properties of TNFα-18ligand prevented it from forming stable oligomeric structures which are essential for effective functioning of the protein. © 2020