Four gelatinolytic proteinases (A1, 97 kDa; A2, 66 kDa; A3, 30 kDa; A4, 23 kDa) were detected from the red stingray wing muscle by gelatin zymography. Among these proteinases, major gelatinolytic proteinases (A1, A2) were more purified and examined their characters. The optimum pH and temperature for the gelatinolytic activity of A1 and A2 were around pH 7 and pH 8, and maximal activity of A1 is at 30–50C, that of A2 is at 30C. Also, they were demonstrated to hydrolyze the gelatin from red stingray; protein bands were almost completely degraded after 6 h incubation at 37C and pH 7.5. Both enzymes were strongly inhibited by Pefabloc SC, which suggests that they belong to gelatinolytic serine protease. PRACTICAL APPLICATIONS Gelatinolytic enzyme hydrolyzes denatured collagen such as gelatin. The enzymes from red stingray may be utilized to produce gelatin peptide, which has been widely used as additive to enhance the elasticity, consistency and stability of food products. In addition, it is meaningful in that this study will give information to understand the gelatinolytic proteinases in ray species and could lead to a comparative study of the biochemical properties of gelatinolytic proteinases in elasmobranch and many other species.