Objective To observe the serum betatrophin levels in patients with type 2 diabetes mellitus (T2DM) and to explore the role of betatrophin in the pathogenesis of diabetic retinopathy (DR). Methods A total of 59 patients with T2DM (DM group) and 14 healthy controls (NC group) were enrolled in the study. Vision, slit lamp microscope, indirect ophthalmoscope, fluorescein fundus angiography were performed on all the subjects. According to the results of the examination combined with the international DR clinical staging criteria, the patients were divided into no DR (Non-DR) group, non-proliferative DR (NPDR) group, and proliferative DR (PDR) group, with 30, 20 and 9 patients in each, respectively. The fasting blood glucose (FPG), insulin (FIN), C-peptide, glycated hemoglobin (HbA1c), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL-C), low-density lipid Protein (LDL-C) levels were detected. The level of betatrophin in serum was determined by enzyme-linked immunosorbent assay. The correlation between betatrophin and other indicators was analyzed by Spearman correlation. The influencing factors of PDR were analyzed by logistic regression. Results Compared with subjects in the NC group, the level of FPG (F=-4.316, P＜0.001), FIN (F=2.142, P=0.001), HbA1c (F=-5.726, P＜0.001), TC (t=3.609, P=0.010), LDL-C (t=0.000, P=0.003), and betatrophin (F=-2.263, P=0.024) were significantly increased and HDL-C level (F=-3.924, P＜0.001) was decreases in the DM group. The difference of TG level between two groups was not statistically significant (F= -1.422, P=0.155). Compared with the Non-DR group and the NPDR group, the serum C-peptide (F=7.818, P=0.020) and betatrophin levels (F=12.141, P=0.002) were significantly increased in the PDR group. Spearman correlation analysis showed that the levels of betatrophin in the DM group was positively correlated to TC (r=0.304, P=0.019). The serum levels of betatrophin was positively correlated to body mass index in the Non-DR group (r=0.513, P=0.004). Furthermore, in the PDR group, a significant positive correlation was observed between the serum betatrophin levels and diastolic blood pressure (r=0.685, P=0.042). Logistic regression analysis showed that the duration of diabetes, serum C-peptide and betatrophin levels were risk factors for PDR. After controlling for the duration and serum C-peptide, the PDR risk for betatrophin levels great than or equal to 1.0 ng/ml was 12 times as much as betatrophin levels less than 1.0 ng/ml in T2DM patients. Conclusions The serum betatrophin content of patients with T2DM is abnormal. Betatrophin may be involved in the occurrence and development of PDR.