Abstract Trypsin-3 is a highly active protease that has recently been identified as a potential therapeutic target for the reduction of tumor growth and metastasis in prostate, breast and pancreatic cancers. Current trypsin inhibitors typically inhibit a broad range of different trypsin-like enzymes. Thus, novel approaches to identify trypsin-3 selective inhibitors are needed. Cyanobacteria produce a vast diversity of natural products with complex chemical structures many of which are potent protease inhibitors. We screened extracts of 140 cyanobacteria strains and discovered numerous strains showing selectivity towards trypsin-2 and -3 inhibition. We isolated a complex glycopeptide from one of these strains, which selectively inhibited human trypsin-2 and -3 with IC50 values of about 100 nM, while trypsin-1 was not inhibited. Importantly, we also found that this peptide inhibited invasion of aggressive and metastatic PC-3M prostate cancer cells though ECM preparation, while it did not affect the proliferation of the cells. Our results suggest that microbial natural products may offer a viable alternative source of potent and selective trypsin-3 inhibitors. Such inhibitors may be suitable, after further development, for targeting the mechanisms associated with the invasion and metastatic dissemination of cancer cells, i.e., for treatment of aggressive cancers. Citation Format: Hannu Koistinen, Matti Wahlsten, Muhammad N. Ahmed, Kaarina Sivonen, Matthias Nees, Ulf-Håkan Stenman, David P. Fewer. Cyanobacterial trypsin-3 inhibitor inhibits prostate cancer cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2679.