The objective of this work was to purify and characterize alkaline protease from Vibrio metschnikovii NG 155 and to study its applicability as a dehairing agent at pilot scale in leather industry. Relative molecular weight of the purified protease was found to be 45 kDa with optimum temperature and pH of 65 °C and 10 respectively. Stability of enzyme in wide range of pH (7–11) and temperature (10–50 °C) makes it suitable for industrial applications. The protease efficiently removed hair from goat skins and buffalo hides, completely eliminating the use of lime and sulphide within short process duration of 8 h. Efficacy of the developed process is supported by histology, scanning electron microscopic analysis and quantification of different skin components. In addition, better physicochemical properties of dyed crusts and lesser pollution load in dehairing process, further affirms the potential of this enzyme for ecofriendly dehairing of animal skins in leather industry.