Liquefaction of human semen involves proteolytic degradation of the seminal coagulum and release of motile spermatozoa. Several members of human kallikrein-related peptidases (KLKs) have been implicated in semen liquefaction, functioning through highly regulated proteolytic cascades. Among these, KLK3 (also known as prostate-specific antigen) is the main executor enzyme responsible for processing of the primary components of semen coagulum, semenogelins I and II. We have recently identified KLK14 as a potential activator of KLK3 and other KLKs. This study aims to elucidate the cascade-mediated role of KLK14 ex vivo. KLK14 expression was significantly lower (p = 0.0252) in individuals with clinically delayed liquefaction. Concordantly, KLK14 expression was significantly (p = 0.0478) lower in asthenospermic cases. Specific inhibition of KLK14 activity by the synthetic inhibitor ACT(G9) resulted in a significant delay in semen liquefaction, a drop in the "early" (30 min postejaculation) "chymotrypsin-like" and KLK1 activity, and an increase in the "late" (90 min postejaculation) chymotrypsin-like activity. Conversely, the addition of recombinant active KLK14 facilitated the liquefaction process, augmented the early chymotrypsin-like activity, and lowered late chymotrypsin-like activity. Given that the observed chymotrypsin-like activity was almost completely attributed to KLK3 activity, KLK3 seems to be regulated bidirectionally. Accordingly, a higher level of KLK3 fragmentation was observed in KLK14-induced coagula, suggesting an inactivation mechanism via internal cleavage. Finally, semenogelins I and II were directly cleaved by KLK14. Semenogelins were also able to reverse KLK14 inhibition by Zn2+, providing a novel regulatory mechanism for KLK14 activity. Our results show that KLK14 exerts a significant and dose-dependent effect in the process of semen liquefaction.