Background: Semenogelin II protein plays a major role in human fertility. Semenogelin II (SGII) is a seminal plasma protein that participates in sperm clotting or gelling in binding with Zn+ ions. Proteolysis of this protein is done by a prostate-specific antigen, and further releases the entrapped sperms after ejaculation. Aim and Objective: To identify the presence of semenogelin II protein in the seminal plasma of various categories and to characterize the protein 3D structure by in silico methods. Methods: Total 137 semen samples have been collected from the Bangalore Assisted Conception Center, Andrology Department, for this research. SDS-PAGE was done to identify the presence of semenogelin II protein. In silico characterization was performed by structural modelling using the ITASSER server. In addition, the structural refinement and protein behaviour were studied by MD simulations using Gromacs. Results: In this research, we identified the presence of semenogelin II in normospermia and oligospermia categories and not in the case of asthenospermia and oligoasthenospermia. This result suggested that the absence of this protein in these categories correlated with motility issues. The structural model of human semenogelin II was generated from the template 4UXV (cytoplasmic domain of the bacterial cell-division protein EzrA) based on the C-score. The predicted model was evaluated by the Ramachandran plot with 86.4% in favoured regions and the overall model quality with a quality index score-3.03, matching with experimental structures. Moreover, MD simulations results showed that the native structure was stable from trajectory analysis. Conclusion: Our study concludes the presence of semenogelin II protein in normospermia and the absence in motility-related infertile categories. Furthermore, the plausible structure of the protein was characterized by in silico methods that can be used for further structure and functional annotation. Thus, semenogellin-II can be a potent marker for the diagnosis of human male infertility. © 2018 Bentham Science Publishers.