Cardiovascular diseases, including ischemic stroke, necessitate improved thrombolytic agents. Staphylokinase (SAK), a microbe-encoded plasminogen activator, is a promising alternative to the widely used tissue plasminogen activator (tPA) due to its high fibrin specificity and low production cost. Yet, its use in clinical settings is hampered by an immunogenicity issue. To overcome this, non-immunogenic variants, SAK-SY155 and SAK-THR174, were engineered. However, the molecular basis underlying their reduced immunogenicity is puzzling and requires detailed elucidation. In this work, we determine molecular structures and compare dynamical behaviour between non-immunogenic and immunogenic SAK variants, using a combination of experimental and computational structural techniques. All variants share the canonical SAK fold and retain similar plasminogen activation kinetics, despite the number of introduced substitutions. Crucially, the non-immunogenic variants exhibit distinct dynamic profiles, with SAK-THR174 showing substantially increased flexibility in the H1 helix and B3 region. Similarly, SAK-SY155 exhibits an increased flexibility in the H1-B3 loop and propensity to homodimerize. These dynamic changes are found in the known immunogenic hotspots. Our multi-scale dynamics analysis thus provides the molecular explanation for the reduced immunogenicity and altered thermostability of the engineered variants, without compromising their fibrinolytic function. This information is critical for the design of next-generation thrombolytics.