The kallikrein-related ( KLK) protease gene family encodes a subgroup of 15 serine proteases that includes prostate-specific antigen (PSA) or KLK3, the well-known biomarker for prostate cancer. PSA is also a major component of seminal fluid. To date, 10 other KLK serine proteases have been documented as present in seminal fluid (KLKs 1, 2, 4, 5, 6, 8, 10, 11, 13, and 14) and, like PSA, have the potential to contribute to male fertility, either directly or indirectly, by means of their proteolytic activity on seminal coagulum proteins. These KLK enzymes arise predominantly from the glandular epithelium of the prostate and are secreted into the lumen of the prostatic ducts that empty into the urethra upon ejaculation. Given their prostatic origin, they are also being considered increasingly as diagnostic/prognostic targets for prostate cancer. This article reviews the literature on seminal fluid PSA and more recent reports on the detection of other KLKs enzymes in this milieu, and their potential roles in male fertility and prostate cancer. We also discuss recent efforts to determine the proteomic profile of seminal fluid to identify new biomarkers for prostate disease.