Abstract: For establishing an efficient in vitro rooting protocol of Camptotheca acuminata and improving the survival and acclimatization of the regenerated plantlets, the effects of auxins, sucrose concentration and different basal media on in vitro rooting and root development at ealier stage after transferred to soil were investigated in this study. We found that, firstly, the addition of auxins to the media can evidently promote rooting. The best rooting was found on the medium containing IBA 0.5 mg.L-1. In this medium, rooting frequencies reached 98%, which associated with a high number of roots, 5.9 roots per shoot. Secondly, root percentage and root number increased with the sucrose concentration from 10 g.L-1 to 30 g.L-1. On the medium containing 30 g.L-1 sucrose, root percentage was 95% with higher root number, 5.4 roots per shoot. Higher sucrose concentration (40 g.L-1) negatively affected rooting. Thirdly, of different medium for rooting, MS medium inhibited root formation. WPM or 1/2MS medium was the optimal medium for in vitro rooting of C. acuminata. Ninety-six precent of the in vitro rooted plantlets with well developed shoots and roots survived after transfer to soil. But root system was much worse than that of seedlings developed from seeds, and the ratio of root tip number to leaf area in regenerated plantlets were significantly lower. Moreover, this ratio was positively correlated with stomatal conductance. The worse root status and low stomatal conductance may be the important reason for the low photosynthetic capacity of regenerated plantlets.