Rodent chronically injected with D-galactose (D-gal) has been used as an animal aging model for brain aging or anti-aging pharmacology research. However, the dosage of D-gal used to establish this model in mice has been reported in a wide range. To study the dose-dependent effect of D-gal on rodent behaviour, we investigated the learning and memory ability of C57BL/6J (C57) mice after 8-week subcutaneous injection of D-gal at different doses by Morris water maze (MWM) and object recognition test (ORT). In addition, locomotor activity test (LAT) was also performed to examine the neuromuscular function. In comparison of vehicle (0.9% saline)-treated mice, D-gal-treated mice at dose of high (200 mg/kg per day) and middle (100 mg/kg per day) doses showed significant longer latency to platform and less target quadrant search time and distance in MWM In ORT, D-gal at high and middle doses reduced the discrimination index (DI) of mice more significantly than low dose (50 mg/kg per day), although all three doses of D-gal reduced the DI of mice significantly. Furthermore, D-gal at high and middle doses significantly decreased locomotor activity of the mice in LAT. Throughout three tests, D-gal induced behavioural impairments in C57 mice at high and middle doses tended to be in the same degree. These results indicate that d-gal can induce the behavioural impairment of C57 mice in a dose-dependent manner from 50 to 100 mg/kg, higher dose than 100 mg/kg cannot further deteriorate its behavioural performance.