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The current study aimed to evaluate the in vitro and in vivo anticancer potential of the silver nanoparticles fabricated with butanol fraction of Pinus roxburghii needles (PNb-AgNPs). In vitro results revealed significant cytotoxicity of PNb-AgNPs towards Ehrlich Ascites Carcinoma (EAC) cells with an IC50 of 47.02 ± 1.11 μg/ml. Further, morphological changes, Reactive Oxygen Species (ROS) generation, mitochondrial depolarization, and DNA fragmentation elucidated EAC cell death by PNbAgNPs exposure. The cell cycle analysis displayed an increase in the Sub-G1 population (52%), further confirmed by nuclear fragmentation in EAC cells after PNb-AgNPs treatment. The hemolytic investigation revealed the biocompatible nature of PNb-AgNPs. In vivo experiments were performed using the liquid Ehrlich Ascites Carcinoma tumor model, which clearly showed a reduction in tumor growth and body weight following PNb-AgNPs treatment in EAC-bearing mice as compared to untreated controls. Moreover, elevated hematological and biochemical parameters were found to restore to normal range in EAC-bearing mice treated with PNb-AgNPs. Overall, PNb-AgNPs effectively induced apoptotic cell death in EAC cells and exhibited remarkable in vivo antitumor potential.