AimsIncreasing numbers of reports link vitamin D deficiency to diabetic peripheral neuropathy (DPN), yet evidence regarding neurological deficits and electromyogram is scarce. The present multi‐centre study sought to investigate these associations based on objective quantifications.Materials and MethodsInformation on DPN‐related symptoms, signs, all diabetic microvascular complications, and nerve conduction abilities (quantified by nerve conduction amplitude and velocity, F‐wave minimum latency (FML) of peripheral nerves) were collected from a derivation cohort of 1192 patients with type 2 diabetes (T2D). Correlation, regression analysis, and restricted cubic splines (RCS) were used to explore linear and non‐linear relationships between vitamin D and DPN, which were validated in an external cohort of 223 patients.ResultsPatients with DPN showed lower levels of vitamin D than those without DPN; patients with vitamin D deficiency (<30 nmol/L) tended to suffer more DPN‐related neurological deficits (paraesthesia, prickling, abnormal temperature, ankle hyporeflexia, and distal pall hypoesthesia correlating with MNSI‐exam score (Y = −0.005306X + 2.105, P = 0.048). Worse nerve conduction abilities (decreased motor nerve amplitude, sensory nerve amplitude, motor nerve velocity, and increased FML) were also observed in these patients. Vitamin D had a significant threshold association with DPN (adjusted OR = 4.136, P = 0.003; RCS P for non‐linearity = 0.003) and correlates with other microvascular complications (diabetic retinopathy and diabetic nephropathy).ConclusionsVitamin D is associated with the conduction ability of peripheral nerves and may have a nerve‐ and threshold‐selective relationship with the prevalence and severity of DPN among patients with T2D.