KISS1 is a broadly functional secreted proprotein that is then processed into small peptides, termed kisspeptins (KP). Since sequence analysis showed cleavage at KR or RR dibasic sites of the nascent protein, it was hypothesized that enzyme(s) belonging to the proprotein convertase family of proteases process KISS1 to generate KP. To this end, cell lines over-expressing KISS1 were treated with the proprotein convertase inhibitors, Dec-RVKR-CMK and α1-PDX, and KISS1 processing was completely inhibited. To identify the specific enzyme(s) responsible for KISS1 processing, mRNA expression was systematically analyzed for six proprotein convertases found in secretory pathways. Consistent expression of the three proteases - furin, PCSK5 and PCSK7 - were potentially implicated in KISS1 processing. However, shRNA-mediated knockdown of furin - but not PCSK5 or PCSK7 - blocked KISS1 processing. Thus, furin appears to be the essential enzyme for the generation of kisspeptins.