Early life stress plays a crucial role in modulation of physiological and psychological processes during ontogenesis. One of the stressors that might contribute to development of mental disorders is maternal separation (MS). The purpose of this study was to evaluate the effect of MS on anxiety of rats during the juvenile transition period. The experiments were performed on both female and male Sprague Dawley rats. The early life repeated MS was proceeded from 11 to 20 postnatal days (PND) in duration of 4 h per day during active phase of circadian rhythm (from ZT15 until ZT19, Zeitgeber time-ZT). Later, at the age of juvenile transition (PND 29), the exploratory and locomotor activity as well as anxiety level were evaluated in an open-field test and an elevated plus-maze test. The behavioural tests were performed during the active period of circadian rhythm (ZT17-22). The results showed that MS increased horizontal locomotor activity and decreased rearing as well as latency to enter into the centre of the open field. On the other hand, animals that were exposed to MS spent less time in the open arm of the elevated plus-maze and expressed less rearing activity. In generally, there were no significant sex differences in the performed tasks. However, female rats exposed to maternal separation had less rearing activity in the elevated plus-maze compared to male rats. The present data indicated that the effect of early life stress caused by MS on exploratory, locomotor and anxiety-like behaviours during early adolescence is task dependent.