Janus kinase is critical for cytokine-mediated signaling, and its hyperactivation due to mutations drives various diseases. The activation of Janus kinase 1 (JAK1) involves a conformational transition from a closed to an open state, but the underlying mechanism remains unclear. This study investigates the roles of two tyrosine residues, Y1034 and Y1035, within the activation loop of the tyrosine kinase domain. Molecular dynamics simulations reveal that phosphorylation, particularly bisphosphorylation at Y1034 and Y1035, promotes the transition to the open conformation, with pY1035 exerting a greater influence than pY1034. Phosphorylation increases the negative charge on the TK domain surface, facilitating its dissociation from the FERM domain, while also weakening TK-FERM interactions. However, the loop between the TK and PK domains formed stable hydrogen bonds with other domains, hindering the full activation process. Using 1 µs molecular dynamics simulations is not sufficient for full activation. These findings elucidate the molecular mechanisms governing the JAK1 initial activation and provide insights for targeting its regulation in disease contexts.