To explore new targets for the prevention and treatment of painful diabetic peripheral neuropathy (DPNPAIN) by comparing and analyzing the clinical and gut microbiota characteristics of patients with DPNPAIN, non-painful diabetic peripheral neuropathy (DPN), and diabetes patients without peripheral nerve injury (NDPN). Methods: Diabetes patients who visited the Department of Neurology or Endocrinology at Beijing Friendship Hospital from January 2021 to December 2022 were screened according to the inclusion and exclusion criteria. They were divided into three groups through medical history inquiry, physical examination, and electrophysiological examination: DPNPAIN group, DPN group, and NDPN group. The basic clinical data of the enrolled patients were analyzed, and stool samples were collected for microbial analysis. Results: A total of 112 patients were enrolled in this study, with 37 cases in the NDPN group, 41 cases in the DPN group, and 34 cases in the DPNPAIN group. Clinical data analysis found that age, eosinophil count, platelet count, high-density lipoprotein, and glycosylated hemoglobin may be related to the occurrence of DPNPAIN. Gut microbiota analysis found that c_Negativicutes and its branching populations were dominant species in the feces of DPNPAIN group patients, which may be biological markers of this disease. Conclusion: This study observed the clinical and gut microbiota characteristics of DPNPAIN through group comparison, speculating that improving the gut microbiota and reducing the content of c_Negativicutes and its branching species could become a new target for the treatment of this disease.