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The aetiology of painful diabetic neuropathy is unclear. We have evaluated vitamin D levels in diabetic patients with and without painful neuropathy. Forty-three patients with type 1 diabetes and painless (DPN) (n = 20) or painful (PDN) (n = 23) neuropathy and 14 non-diabetic healthy control subjects (C) underwent assessment of neurologic deficits, quantitative sensory testing (QST), electrophysiology, skin biopsy, corneal confocal microscopy (CCM) and measurement of serum 25(OH)D. There were no significant differences for age, BMI, HbA1c, lipids, neurological deficits, QST, electrophysiology, intra-epidermal nerve fibre density (IENFD) and corneal nerve morphology between patients with DPN and PDN. Both positive (hyperalgesia and allodynia) and negative symptoms (paraesthesia and numbness) of diabetic neuropathy were greater in PDN compared to DPN (P = 0.009 and P = 0.02 respectively). Serum 25(OH)D level were significantly lower in PDN (24.0 ± 14.1 ng/mL) compared to DPN (34.6 ± 15.0 ng/mL, P = 0.01) and controls (34.1 ± 8.6 ng/mL, P = 0.03). The odds ratio in favour of painful diabetic neuropathy was 9.8 (P = 0.003 (95% CI 2.2-76.4) for vitamin D deficiency (<20 ng/mL) and 4.4 (P = 0.03 [95% CI 1.1-19.8]) for vitamin D insufficiency (<30 ng/mL). This study suggests that vitamin D deficiency and insufficiency are associated with painful diabetic neuropathy. This article is protected by copyright. All rights reserved.