Halolysins are extracellular proteases secreted by halophilic archaea for nutritional purposes. They bear great application potentials in various industries. Yet the diversity of halolysins remains underexplored. In this study, a halolysin from the extremely halophilic archaeon Haladaptatus sp. DYF46 (Hly Hap ) was identified to be a novel type of halolysin without C-terminal extension (CTE). Addition of the CTE of a halolysin from Halococcus salifodinae to Hly Hap did not significantly affect its extracellular proteolytic activity. Mature Hly Hap was generated from recombinant Hly Hap precursor by high-affinity column refolding. Hly Hap displayed optimal activity at 0.25-0.50 M NaCl, 45 °C and pH 8.5-9.0. Interestingly, Hly Hap preferred a low salinity and was stable in a broad range of salinity, albeit from an extremely halophilic archaeon. Ca 2+ and Mg 2+ significantly promoted Hly Hap activity. Hly Hap activity was stable with organic solvents and detergents. The K m and V max values of Hly Hap against azocasein were 0.018 mM and 7,179 U/mg, and those against succinyl-Ala-Ala-Pro-Phe-pNA were 0.32 mM and 3×10 6 μmol/min/μg, respectively. The unusual traits of Hly Hap , a novel type of halolysin without CTE, may endow it with strong potential for various industrial uses, such as biocatalysis in fluctuating salinities and aqueous-organic solvent. Key points • This is the first report of a novel type of halolysin without C-terminal extension • Hly Hap was obtained by heterologous expression and high-affinity column refolding • Hly Hap exhibited good salinity tolerance