Objective To assess the effect of high-dose vitamin D in patients with painful diabetic neuropathy. Methods A single intramuscular dose of 600 000 IU vitamin D was administered, and the effects on metabolic parameters and neuropathic pain assessed over 20 weeks. Results 143 participants with predominantly type 2 diabetes, aged 52.31±11.48 years, with a Douleur Neuropathique 4 (DN4) score (3.0±1.8), total McGill pain score (21.2±14.9), and Short Form McGill Pain Questionnaire (SFMPQ) score (2.1±0.9), were enrolled. The baseline 25-hydroxyvitamin D (25(OH)D) level was 31.7±23.3 ng/mL and 58 (40.5%) patients showed evidence of vitamin D deficiency (25(OH)D<20 ng/mL). Intramuscular administration of vitamin D resulted in a significant increase in 25(OH)D (46.2±10.2 ng/mL, p<0.0001) and a reduction in positive symptoms on the DN4 (p<0.0001), total pain score (p<0.0001), and SFMPQ (p<0.0001). Conclusions Treatment with a single intramuscular dose of 600 000 IU of vitamin D in patients with painful diabetic neuropathy is associated with a significant decrease in the symptoms of painful diabetic neuropathy. Trial registration number BIDE-12/2014.