Seminal plasma proteins of the fibronectin type II (Fn2) family modulate mammalian spermatogenesis by triggering the release of the lipids phosphatidylcholine and cholesterol from sperm cells. Whereas the specific interaction of these proteins with phosphatidylcholine is well-understood, their selectivity for cholesterol is unknown. To characterize the interaction between the bovine Fn2 protein PDC-109 and cholesterol, we have investigated the effect of PDC-109 on the dynamics of fluorescent cholesterol analogues in lipid vesicles by time-resolved fluorescence anisotropy. The data show that PDC-109 decreases the rotational mobility of cholesterol within the membrane and that the extent of this impact depends on the cholesterol structure, indicating a specific influence of PDC-109 on cholesterol. We propose that the cholesterol recognition/interaction amino acid consensus (CRAC) regions of PDC-109 are involved in the interaction with cholesterol.