Abstract: To observe the effects of glycerol on energy balance, blood parameter, glucose and adipose concentrations in the liver during the peripartum period in dairy cows, multiparous Holstein dairy cows with similar body weights (674.6±13.8 kg), lactation numbers (2.5±0.5), corrected milk yields in the last lactation (7 412±15 kg) and expected calving dates (23.8±0.3 d) were allocated into four treatments at random. Graded levels (0, 100, 200, and 300 g/d) of glycerol were supplemented in the four treatments from day 19 peripartum. Body condition score at 7, 21 and 35 d postpartum was increased, body weight loss from 7 to 35 d postpartum was reduced, energy balance at 14 d peripartum and 7, 21 and 35 d postpartum was improved significantly (P<0.05) by the 200 g/d and 300 g/d treatment compared with the 0 g/d treatment. Cows receiving 200 g/d and 300 g/d glycerol had a higher insulin concentration at 14 d peripartum and higher plasma glucose and insulin concentrations at 7, 21 and 35 d postpartum than the 0 g/d treatment (P<0.05). They had lower plasma NEFA and β-hydroxybutyrate at 7, 21 and 35 d postpartum than the 0 g/d and 100 g/d treatment (P<0.05) while 200 g/d and 300 g/d glycerol supplementation increased the contents of glycogen and key regulatory enzymes of gluconeogenesis in the liver compared with the 0 g/d glycerol treatment (P<0.05). Glycerol supplementation was beneficial to peripartum cows in enhancing the gluconeogenesis and improving energy balance status. The optimum dose was about 200 g per cow per day.