SP-D an innate immune molecule has indispensable role in host defense and regulation of inflammation. We reported a novel anti-cancer role of SP-D in various cancer cell lines. In accordance with our previous finding, rfhSP-D induced intrinsic mitochondrial apoptosis in LNCaP (androgen-dependent) cells, PC3 (androgen-independent) cells, human prostate tissue biopsies and primary cancer cells (PrCEC) isolated from tissue biopsies of metastatic prostate cancer patients, in time and dose-dependent manner. Importantly, viability of primary normal prostate epithelial cells (PrEC) was not affected. In the present study, we observed significantly higher calcium dependent binding of rfhSP-D with the PC3 cells than LNCaP and PrEC cells suggesting the differential expression of interacting proteome. LC-MS/MS analysis of rfhSP-D-PC3 interactome resulted in identification of 672 proteins. Further screening of these proteins based on the abundance (proteins with high Peptide Spectrum Match) and molecular function resulted a list of 25 potential interacting partners. PANTHER analysis suggested that these proteins are part of Apoptosis signaling pathway, Glycolysis, Gonadotropin- releasing hormone receptor pathway and Integrin signaling pathway. We selected GRP78, a chaperone, expressed only on cell surface of cancer cells, and involved in apoptosis signaling pathway for further analysis. The structures of active rfhSP-D (PDB ID: 1PW9) in the trimeric form and GRP78 (PDB ID: 5EVZ) were docked using PatchDock and ZDock server with default parameters. Thus, the proteomics and insilico approach confirmed interaction of membrane GRP78 protein with carbohydrate recognition domain of rfhSP-D on prostate cancer cells, and may be involved in anti-prostate cancer role of SP-D.