Abstract Background Spike protein is a key player in the SARS-CoV-2 infection by mediating primary contact between the virus and host cell surface. In the current COVID-19 pandemic, a variant of SARS-CoV-2 having D614G substitution in the spike protein has become dominant world-wide. Initial characterization of the virus shows that the G614 variant is more infectious and has higher fitness than the ancestral (D614) variant. In this study, we analyzed the significance of the D614G substitution on the protein flexibility, inter-residue interaction energies and thermostability of the spike protein trimer. Results Using Gaussian network model-based normal mode analysis, we demonstrate that D614G substitution occurs at hinge region that facilitates domain-domain motions between receptor binding domain and S2 region of the spike protein. Further, in-silico mutagenesis and inter-residue energy calculations reveal that contacts involving D614 are energetically frustrated whereas contacts involving G614 are energetically favourable implying the substitution strengthens intra- as well as inter-protomers association. Upon glycine substitution, free energy difference (ΔΔG) is -2.6 kcal/mol for closed and − 2.0 kcal/mol for 1-RBD up conformation i.e., thermodynamic stability has increased. When we perform reverse mutation in the structures of spike protein having G614 substitution, we observe that the free energy difference is 6.6 kcal/mol and 6.3 kcal/mol for closed and 1-RBD up conformations respectively indicating lowered thermodynamic stability. Together, these observations suggest that D614G substitution could modulate the flexibility of spike protein and confer enhanced thermodynamic stability. Conclusion Our results on protein flexibility and energetic basis of enhanced stability hint that G614 likely increases the availability of functional form of spike trimer thereby associated to increased infectivity.