The present study was aimed at the investigation of the effects of melatonin on spinal cord injury (SCI) and the role of IGFBP3 in SCI both in vivo and in vitro. The rats received treatment with 100 mg kg⁻¹ melatonin or both melatonin and pGenesil-1-si-IGFBP3 (50 µg per g bw) after SCI surgery. The motor function in rats was measured using the Basso–Beattie–Bresnahan (BBB) scale score; perfusion vessel area was determined by injecting FITC-conjugated lycopersicon esculentum agglutinin lectin (FITC-LEA), whereas the blood–spinal cord barrier permeability was measured using Evans blue. The pericytes were isolated, and the cells were cultured under hypoxia, treated with melatonin or transfected with si-IGFBP3. RT-qPCR and western blotting were conducted for the determination of IGFBP3, VEGF, MMP-2, ICAM-1 and Ang1. The expression of IGFBP3 was significantly down-regulated in the SCI rats, and melatonin significantly enhanced the IGFBP3 level. Melatonin improved the motor function, reduced the neuron injury, and improved the microcirculation in rats. However, the down-regulation of IGFBP3 significantly reversed these effects. Moreover, in both the SCI rat spinal cord tissues and the in vitro pericytes under hypoxia, the expressions of IGFBP3 and Ang1 were significantly down-regulated, whereas those of the proteins MMP-2, VEGF and ICAM-1 were significantly up-regulated, and melatonin dramatically inhibited these changes. Melatonin could protect the rats from SCI by improving the microcirculation through the up-regulation of IGFBP3.