There are sporadic reports that assorted combinations of B vitamins can alleviate pain in diabetic patients, but there is neither agreement on the relative efficacy of individual B vitamins nor understanding of the mechanisms involved. We therefore investigated the efficacy of a cocktail of the vitamins B1, B6 and B12 in alleviating behavioral indices of sensory dysfunction such as allodynia and hyperalgesia in diabetic rats and also the relative contribution of individual components of the cocktail. Repeated daily treatment with the cocktail of B vitamins for 7–9 days ameliorated tactile allodynia and formalin-evoked hyperalgesia in a dose-dependent manner and also improved sensory nerve conduction velocity in diabetic rats. Investigation of the contribution of individual B vitamins suggested that all three participated with variable efficacy in the alleviation of allodynia after protracted, but not single dose treatment. Only vitamin B6 improved sensory nerve conduction velocity slowing in diabetic rats when given alone. To address potential mechanisms of action, we measured markers of oxidative stress (lipid and protein oxidation) and inflammation (cyclooxygenase-2 (COX-2) and TNFα protein) in the nerve but treatment with the vitamin B cocktail did not significantly affect any of these parameters. The positive effects of B vitamins on functional and behavioral disorders of diabetic rats suggest a potential for use in treating painful diabetic neuropathy.