HSPA2, a testis-specific protein belonging to the HSP70 family, is ubiquitously found in the testes and spermatozoa. It plays a pivotal role in the spermatogenesis process, and associated with human sperm-egg recognition. During various stress conditions, ligand-receptor binding between extracellular HSP70 and TLR 4 has been well established in many cells, except spermatozoa. Bacteriospermia-associated leukocytospermia (LCS/BS)-induced immune infertility is a pathophysiological condition of the male reproductive system mediated by antisperm antibody (ASA) production and TLR 4 signaling. In this study, the mechanisms involved in the interaction between HSPA2 and TLR 4 in the spermatozoa were revealed. Based on previously published data which revealed the identification of several types of seminal bacteria from the LCS/BS patients attending Kar Clinic and Hospitals Pvt. Ltd., semen samples containing Escherichia coli infection were selected for this particular study. The immunocytochemistry study showed co-localization of HSPA2 with TLR 4 on the spermatozoa surface confirming a plausible interaction between them. The HSPA2-TLR 4 interaction was further strengthened by multiple pathway analytical tools such as Cytoscape PSICQUIC client service and STRING database version 11.0. Upon CLUSTAL Ω analysis, DnaK and HSPA2 protein sequences showed 47% identity. Using IEDB tool it was revealed that thirty-eight self-peptides of E. coli DnaK protein shared homology with HSPA2. This was further confirmed by the strong binding affinity between MHC class-II DRB allele and DnaK/HSPA2 as evidenced by PATCHDOCK software. Altogether, a true interaction between HSPA2 and TLR 4 signaling on the spermatozoa surface was deciphered via dual mechanisms: (1) the direct interaction due to LCS/BS-induced immune infertility; (2) and the indirect one where, HSPA2 is the mimicked version of DnaK protein isolated from E. coli. These findings indicate the existence of a ligand-receptor relationship between HSPA2 and TLR 4 on the spermatozoa surface during immune infertility.