Objective To investigate the level of serum homocysteine (Hcy) in patients with type 2 diabetes mellitus (T2DM) accompanied with diabetic peripheral neuropathy (DPN) and the relationship between Hcy and diabetic nephropathy (DN), and to explore the mechanisms of Hcy in T2DM patients with DPN. Methods   38 patients with T2DM accompanied with DPN (DPN group), 35 T2DM patients without DPN (NDPN group), and 32 normal controls were chosen (NC group) in this study. Fasting serum homocysteine (Hcy) level was measured by enzymatic cycling assay and compared. The relationship in the change of Hcy, cystatin C (Cys-C), microalbuminuria-to-creatinine ratio (ACR), glomerular filtration rate (GFR), blood pressure, glucose and blood lipid and other parameters was also analyzed. Results Compared with those of control group, serum Hcy level in patients with DPN and NDPN groups significantly increased (15.13±1.56 vs. 10.77±3.59 vs. 7.19±1.95μmol/L, P<0.05 or P<0.01).Moreover, DPN patients were detected with higher serum Hcy levels than NDPN patients (15.13±1.56 vs. 10.77±3.59μmol/L, P<0.05). Serum Hcy levels correlated negatively with GFR, and  correlated positively with vibration perception threshold (VPT), serum creatinine (Scr), Cys-C and ACR in DPN patients (P<0.05 or P<0.01). Multiple regression analysis showed that Cys-c and VPT were independent related factors influencing Serum Hcy levels. Conclusions Serum Hcy levels are significantly increased in T2DM patients, especially for DPN patients, which are significantly correlated with early renal function of diabetic nephropathy and may play an important role in the occurrence and development of DPN in T2DM patients.