AbstractThis phase Ib, non‐randomized, open‐label study evaluates the safety and tolerability of ruxolitinib in combination with nilotinib and prednisone in patients with naïve or ruxolitinib‐resistant myelofibrosis (MF). A total of 15 patients with primary or secondary MF received the study treatment; 13 patients had received prior ruxolitinib treatment (86.7%). Eight patients completed seven cycles (53.3%) and six patients completed twelve cycles of treatment (40%). All the patients experienced at least one adverse event (AE) during the study (the most common AEs were hyperglycemia, asthenia, and thrombocytopenia), and 14 patients registered at least one treatment‐related AE (the most common treatment‐related AEs were hyperglycemia (22.2%; three grade 3 cases). Five treatment‐related serious AEs (SAEs) were reported in two patients (13.3%). No deaths were registered throughout the study. No dose‐limiting toxicity was observed. Four out of fifteen (27%) patients experienced a 100% spleen size reduction at Cycle 7, and two additional patients achieved a >50% spleen size reduction, representing an overall response rate of 40% at Cycle 7. In conclusion, the tolerability of this combination was acceptable, and hyperglycemia was the most frequent treatment‐related AE. Ruxolitinib in combination with nilotinib and prednisone showed relevant clinical activity in patients with MF. This trial was registered with EudraCT Number 2016‐005214‐21.