Silicon (Si) nanostructures are widely used in microelectronics and nanotechnology. Brittle to ductile transition in nanoscale Si is of great scientific and technological interest but this phenomenon and its underlying mechanism remain elusive. By conducting in situ temperature-controlled nanomechanical testing inside a transmission electron microscope (TEM), here we show that the crystalline Si nanowires under tension are brittle at room temperature but exhibit ductile behavior with dislocation-mediated plasticity at elevated temperatures. We find that reducing the nanowire diameter promotes the dislocation-mediated responses, as shown by 78 Si nanowires tested between room temperature and 600 K. In situ high-resolution TEM imaging and atomistic reaction pathway modeling reveal that the unconventional 1/2⟨110⟩{001} dislocations become highly active with increasing temperature and thus play a critical role in the formation of deformation bands, leading to transition from brittle fracture to dislocation-mediated failure in Si nanowires at elevated temperatures. This study provides quantitative characterization and mechanistic insight for the brittle to ductile transition in Si nanostructures.