Ovarian cancer is a leading cause of cancer death as diagnosis is frequently delayed to an advanced stage. Effective biomarkers and screening strategies for early detection are urgently needed. In the current study, we identify PSP94 as a key upstream factor in mediating prostasin (a protein previously reported to be overexpressed in ovarian cancer) signaling that regulates prostasin expression and action in ovarian cancer cells. PSP94 is overexpressed in ovarian cancer cell lines and patients, and is significantly correlated with prostasin levels. Signaling pathway analysis demonstrated that both PSP94 and prostasin, as potential upstream regulators of the Lin28b/Let-7 pathway, regulate Lin28b and its downstream partner Let-7 in ovarian cancer cells. Expression of PSP94 and prostasin show a strong correlation with the expression levels of Lin28b/Let-7 in ovarian cancer patients. Thus, PSP94/prostasin axis appears to be linked to the Lin28b/Let-7 loop, a well-known signaling mechanism in oncogenesis in general that is also altered in ovarian cancer. The findings suggest that PSP94 and PSP94/prostasin axis are key factors and potential therapeutic targets or early biomarkers for ovarian cancer.