Molecular dynamics (MD) at two temperatures of 300 and 340 K identified two histidine residues, His461 and His489, in the most flexible regions of firefly luciferase, a light emitting enzyme. We therefore designed four protein mutants H461D, H489K, H489D and H489M to investigate their enzyme kinetic and thermodynamic stability changes. Substitution of His461 by aspartate (H461D) decreased ATP binding affinity, reduced the melting temperature of protein by around 25 °C and shifted its optimum temperature of activity to 10 °C. In line with the common feature of psychrophilic enzymes, the MD data showed that the overall flexibility of H461D was relatively high at low temperature, probably due to a decrease in the number of salt bridges around the mutation site. On the other hand, substitution of His489 by aspartate (H489D) introduced a new salt bridge between the C-terminal and N-terminal domains and increased protein rigidity but only slightly improved its thermal stability. Similar changes were observed for H489K and, to a lesser degree, H489M mutations. Based on our results we conclude that the MD simulation-based rational substitution of histidines by salt-bridge forming residues can modulate conformational dynamics in luciferase and shift its optimal temperature activity. Display omitted •In this research, molecular dynamics (MD) simulation was carried out to assess the molecular stability and flexibility of luciferase enzyme.•Mutations of Histidine in the flexible region altered salt bridges and hydrogen bonding propensities and changed enzyme kinetic parameters and thermodynamic stability.•H461D reduced the melting point of protein and shifted its optimum temperature of activity to lower temperature like as psychrophilic enzymes.•In medical and industry procedures whenever requires to do reactions with low energy consumption and cold temperature environment, this psychrophilic luciferase can be potentially utilized.