Objective To observe the changes of neuronal apoptosis, learning and memory, and the effects of drug accumulation after repeated use of chloral hydrate (CHL) or dexmedetomidine (Dex) in neonatal rats. Methods Seventy‑eight neonatal SD rats were assigned to 4 different sedation groups using random number table method: chloral hydrate group (CHL group, n=19), dexmedetomidine group (Dex group, n=19), drug mixed group (C+D group, n=20) and normal saline group (NS group, n=20, placebo). From the 8th day of birth, sedative doses of CHL (40 mg/kg), Dex (10 μg/kg), C+D (CHL 20 mg/kg and Dex 5 μg/kg), or equal amount of NS were injected intraperitoneally for 4 consecutive days respectively. Three rats in each group were randomly sacrificed by decapitation on the day after each injection and brain tissue samples were taken. The caspase‑3 and cleaved caspase‑3 in brain tissue from one hemisphere was detected by Western blot (gelatin electrophoresis) after all brain tissues were collected. The brain tissue from another hemisphere was fixed in 4% paraformaldehyde (PFA), frozen sections were prepared, and terminal deoxynucleotidyl transferase‑imdiated dUTP‑biotin mick end labeling (TUNEL) method was used for apoptosis observation. The remaining rats were raised to 7 to 8 weeks old for Morris water‑maze test, in which 5 d navigation training and 60 s space exploration experiment were conducted to evaluate the learning and memory ability. Results Brain tissue apoptosis detection: Western blot showed that cleaved caspase‑3 bands were not obvious at all time points in each group, and no apoptotic bodies were observed by TUNEL method in frozen sections. In Morris water‑maze test, there was no significant difference in the time of reaching the target platform for the rats in each group (P>0.05). Compared with the NS group and Dex group, the time of first crossing platform area in CHL group was longer and the number of rats which first crossed the platform area in the same time was decreased, but the difference was not statistically significant (P>0.05). Conclusions The normal sedative dose of CHL in this study showed no effect on the apoptosis, structure and learning ability of brain cells in neonatal SD rats, but the spatial memory function was damaged, and the above effects had no cumulative effect.