Diabetic retinopathy (DR) is a common complication of diabetes mellitus. High glucose‐induced mitochondrial apoptosis is involved in the loss of retinal pericytes (PCs), which is considered to be a predominant pathologic change of diabetic retinopathy (DR). A high thyroid stimulating hormone (TSH) serum level is associated with an increased prevalence of DR in diabetic patients. Here, we investigated whether TSH regulated glucose‐induced PCs loss through TSH‐receptor (TSHR)‐dependent mitochondrial apoptosis. First, the serum TSH level was found to be an independent risk factor for DR in Type 2 diabetic study participants (odds ratio = 2.294; 95% confidence interval: 1.925–2.733; p ≤ 0.001). Second, human PCs were treated with different concentrations of glucose, with or without bovine TSH (b‐TSH). Glucose induced mitochondrial apoptosis through various mechanisms, including through regulating the expression of apoptosis‐related proteins and inducing mitochondrial dysfunction, which could be deteriorated by costimulation of glucose and b‐TSH. Additionally, we detected functional TSHR in PCs; blocking TSHR significantly restricted TSH‐induced apoptosis. Thus, the presence of functional TSHR in human retinal PCs may facilitate the effect of high TSH on high glucose‐induced PCs loss through TSHR‐dependent mitochondrial apoptosis.