We conducted a systematic review and meta-analysis to evaluate the effect of Berberine on glucose in patients with type 2 diabetes mellitus and identify potential factors may modifying the hypoglycemic effect. We searched PubMed, Embase, the Cochrane Library, China National Knowledge Infrastructure, and Wanfang Database to identify randomized controlled trials that investigated the effect of Berberine. We calculated weighted mean differences (WMD) and 95% confidence interval (CI) for fasting plasma glucose (FPG), postprandial plasma glucose (PPG) and glycated haemoglobin (HbA1c) levels. Twenty-eight studies were identified for analysis, with a total of 2,313 type 2 diabetes mellitus (T2DM) patients. The pool data showed that Berberine treatment was associated with a better reduction on FPG (WMD = –0.54 mmol/L, 95% CI: –0.77 to –0.30), PPG (WMD = –0.94 mmol/L, 95% CI: –1.27 to –0.61), and HbA1c (WMD = –0.54 mmol/L, 95% CI: –0.93 to –0.15) than control groups. Subgroup-analyses indicated that effects of Berberine on blood glucose became unremarkable as the treatment lasted more than 90 days, the daily dosage more than 2 g/d and patients aged more than 60 years. The efficiency of Berberine combined with hypoglycaemics is better than either Berberine or hypoglycaemic alone. The dosage and treatment duration of Berberine and patients’ age may modify the effect.