Properties of film from cuttlefish (Sepia pharaonis) ventral skin gelatin with different degree of hydrolysis (DH: 0.40, 0.80 and 1.20%) added with glycerol as plasticizer at various levels (10, 15 and 20%, based on protein) were investigated. Films prepared from gelatin with all DH had the lower tensile strength (TS) and elongation at break (EAB) but higher water vapor permeability (WVP), compared with the control film (without hydrolysis) (p<0.05). At the same glycerol content, both TS and EAB decreased, while WVP increased (p<0.05) with increasing %DH. At the same DH, TS generally decreased as glycerol content increased (p<0.05), however glycerol content had no effect on EAB when gelatins with 0.80 and 1.20% DH were used (p>0.05). DH and glycerol content had no marked impact on color and the difference in color (ΔE ∗ ) of resulting films. Electrophoretic study revealed that degradation of gelatin and their corresponding films was more pronounced with increased %DH, resulting in the lower mechanical properties of films. Based on FTIR spectra, with the increasing %DH as well as glycerol content, higher amplitudes for amide-A and amide-B peaks were observed, compared with film from gelatin without hydrolysis (control film) due to the increased –NH 2 group caused by hydrolysis and the lower interaction of –NH 2 group in the presence of higher glycerol. Thermo-gravimetric analysis indicated that film prepared from gelatin with 1.20% DH exhibited the higher heat susceptibility and weight loss in the temperature range of 50–600°C, compared with control film. Thus, both chain length of gelatin and glycerol content directly affected the properties of cuttlefish skin gelatin films.