The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109.