Agents used to treat symptoms of diabetic peripheral neuropathy (DPN) are only palliative, not disease modifying. Although studies of monotherapy with L-methylfolate, methylcobalamin, or pyridoxal 5'-phosphate suggest that each of these bioavailable B vitamins may reverse the pathophysiology and symptoms of DPN, data on the efficacy of this combination therapy are limited. Therefore, we assessed the efficacy of an oral combination of L-methylfolate, methylcobalamin, and pyridoxal 5'-phosphate for improving epidermal nerve fiber density (ENFD) in the lower extremity of patients with DPN. Eleven consecutive patients with type 2 diabetes with symptomatic DPN were assessed for ENFD at the calf by means of skin punch biopsy and then placed on twice daily oral-combination L-methylfolate, methylcobalamin, and pyridoxal 5'-phosphate. After approximately 6 months of treatment, patients underwent follow-up biopsy. At the end of their treatment, 73% of patients showed an increase in calf ENFD, and 82% of patients experienced both reduced frequency and intensity of paresthesias and/or dysesthesias. This preliminary study suggests that combination L-methylfolate, methylcobalamin, and pyridoxal 5'-phosphate increases ENFD in patients with DPN.