Objective To evaluate the effect of maternal separation stress on the behavior of neonatal rd mice. Methods Neonatal rd mice were divided into maternal separation (MS) group (n=9) and control group (n=9). MS-stress was induced in the MS group by 4-hour-separation per day for 28 days. Open field test, elevated plus maze test, forced swim test and tail suspension test were used to evaluate the anxiety-like and depression-like behavior of the neonatal rd mice.Results The stay time and distance travelled of MS group in the central zone were 0.88% and 28.17±5.65 cm, respectively, significantly shorter than that of the control group (2.61%,109.9±9.79 cm. P=0.04, P=0.001). Compared with the control group, the stay time in open arms of the MS group was significantly decreased (P<0.01), while the immobility time in forced swim test and tail suspension test of the MS group were 126.5±10.22 s and 21.56±6.83 s, significantly longer than that of the control group (77.75±16.83 s, P=0.02, 7.37±3.22 s, P=0.03). Conclusions The 28-day maternal separation stress can significantly increase the anxiety-like and depression-like behavior in neonatal rd mice.