The characterisation of a serine protease isolated from tamarillo (Solanum betaceum) fruit and its milk casein hydrolysis activity were investigated. Compared with calf rennet, a crude extract from tamarillo exhibited wider caseinolytic activity on sodium caseinate. The purified protease was named "tamarillin" and revealed proteolytic activity toward purified α-, β- and κ-casein. Similar to calf rennet, tamarillin preferably hydrolysed κ-casein, but, unlike calf rennet, it also displayed high proteolytic activity toward both α- and β-casein. The major peptide generated from κ-casein by tamarillin was analysed by gel electrophoresis and liquid chromatography mass spectrometry to confirm its molecular mass as 14,290 Da. The cleavage site was confirmed by in-gel tryptic digestion and time-of-flight mass spectrometry analysis to be at Asn123-Thr124. This was in contrast to the Phe105-Met106 cleavage site of rennet hydrolysis.