Chemotherapy remains the main approach conserving vision during the treatment of retinoblastoma, the most prevalent eye cancer in children. Unfortunately, the development of chemoresistance stands as the primary reason for treatment failure. Within this study, we showed that prolonged exposure to vincristine led to heightened expression of JAK1 and JAK2 in retinoblastoma cells, while the other members of the JAK family exhibited no such changes. Employing a genetic intervention, we demonstrated the efficacy of depleting either JAK1 or JAK2 in countering vincristine-resistant retinoblastoma cells. In addition, the dual depletion of both JAK1 and JAK2 produced a more potent inhibitory outcome compared to the depletion of either gene alone. We further demonstrated that ruxolitinib, a small molecular inhibitor of JAK1/2, effectively reduced viability and colony formation in vincristine-resistant retinoblastoma cells. It also acts synergistically with vincristine in retinoblastoma cells regardless of inherent cellular and genetic heterogeneity. The effectiveness of ruxolitinib as standalone treatment against chemoresistant retinoblastoma, as well as its combination with vincristine, was validated in multiple retinoblastoma mouse models. Importantly, mice exhibited favorable tolerance to ruxolitinib administration. We confirmed that the underlying mechanism of ruxolitinib's action in chemoresistant retinoblastoma cells is the inhibition of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. Our study reveals that the underlying mechanism driving ruxolitinib's impact on chemoresistant retinoblastoma cells is the inhibition of JAK/STAT signaling. This study reveals the contribution of JAK1/2 to the development of chemoresistance in retinoblastoma and underscores the effectiveness of targeting JAK1/2 as a strategy to sensitize retinoblastoma to chemotherapy.Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.