Abstract Context About one-third of diabetic patients suffer from neuropathic pain, which is poorly responsive to analgesic therapy and associated with greater autonomic dysfunction. Previous research on diabetic neuropathy mainly links pain and autonomic dysfunction to peripheral nerve degeneration resulting from systemic metabolic disturbances, but maladaptive plasticity in the central pain and autonomic systems following peripheral nerve injury has been relatively ignored. Objective This study aimed to investigate how the brain is affected in painful diabetic neuropathy (PDN), in terms of altered structural connectivity (SC) of the thalamus and hypothalamus that are key regions modulating nociceptive and autonomic responses. Methods We recruited 25 PDN and 13 painless (PLDN) diabetic neuropathy patients, and 27 healthy adults as controls. The SC of the thalamus and hypothalamus with limbic regions mediating nociceptive and autonomic responses was assessed using diffusion tractography. Results The PDN patients had significantly lower thalamic and hypothalamic SC of the right amygdala compared with the PLDN and control groups. In addition, lower thalamic SC of the insula was associated with more severe peripheral nerve degeneration, and lower hypothalamic SC of the anterior cingulate cortex was associated with greater autonomic dysfunction manifested by decreased heart rate variability. Conclusion Our findings indicate that alterations in brain structural connectivity could be a form of maladaptive plasticity after peripheral nerve injury, and also demonstrate a pathophysiological association between disconnection of the limbic circuitry and pain and autonomic dysfunction in diabetes.