The long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition receptor, involved in several processes ranging from innate resistance and inflammation to clearance of apoptotic cells and organization of hyaluronic acid-rich extracellular matrices. PTX3 is also a novel marker in several pathological conditions of infectious, inflammatory, or autoimmune origin. This study was designed to assess whether PTX3 is expressed in the male reproductive tract and whether PTX3 interacts with human spermatozoa influencing their function. Here we show for the first time by immunohistochemistry that PTX3 is expressed in the male genital tract in perivascular connective tissue, in endothelial cells, in the interstitium, and in the cytoplasm of prostatic epithelial glandular cells; PTX3 was detectable in seminal plasma in variable levels, which correlated with the percentage of normal spermatozoa. Moreover, PTX3 binds to spermatozoa, in particular with immotile cells, localizing in the neck and in the subacrosomial region. Finally, recombinant PTX3 did not interfere with sperm motility.