Objective This study aimed to observe the protective effects of 17β-estradiol (17β-E2) to the damage of phenobarbital (PB) upon the cognition of the newly-born rats. Methods Thirty healthy 3-day-old Sprague Dawley (SD) rats were randomly divided into 3 groups: control group (10 rats), PB group (10 rats) and PB+17β-E2 group (10 rats). The control group were injected saline water with a dose of 10 mL/(kg·d); the PB group were injected with PB of 10 mg/(kg·d); the PB+17β-E2 group were injected with PB 10 mg/(kg·d) and 17β-E2 300 ug/(kg·d). All the rats were intraperitoneal injected once a day after weighing, for three continuous days. They were normally raised to one month old and then 8 rats were selected out of each group respectively for water maze test. Results The PB group was reported to have increasing latent periods in finding the underwater stage compared with control group (P<0.05). In comparison with the PB group, the PB+17β-E2 group has a shorter latent period in finding the underwater stage, and furthermore statistically significantly fewer times in finding validation areas (P<0.05). During the 120s test, the stage quadrant journey to total journey ratio of PB+17β-E2 group was lower than the ratio of PB groups, but no statistics significance had been detected. The PB+17β-E2 group exhibits no significance difference from the control group in the above-mentioned indexes. Conclusions Even a short-term injection of PB with an usual clinical dose will bring a long-term damage to an immature brain in terms of the learning ability and memory, whereas the 17β-E2 may play a protective role in this course.