Rooting ability of rice seedling for mechanical transplanting has a large impact on grain yield. This study explored the relationship between endogenous soluble sugar content and rooting ability of rice seedlings. We placed 15-day-old rice seedlings in controlled environment cabinets with stable light and sampled after 0, 3, 6, 9, 12, and 24 hours of light to measure their soluble sugar content, nitrate content, starch content, soluble protein content and rooting ability. The soluble sugar content of the rice seedlings before rooting increased rapidly from 65.1 mg g-1 to 126.3 mg g-1 in the first 9 hours of light and then tended to stabilize; however, few significant changes in the other physiological indices were detected. With the light exposure time increasing from 3 hours to 12 hours, the rooting ability measured with fresh weight, dry weight, total length, and number of new roots increased by 91.7%, 120.0%, 60.6% and 30.3%, respectively. Rooting ability was related more closely to soluble sugar content than to nitrate-nitrogen content of rice seedlings before rooting and their correlation coefficients were 0.8582-0.8684 and 0.7045-0.7882, respectively. The stepwise regression analysis revealed that the soluble sugar content before rooting explained 73.6%-75.4% of the variance, and the nitrate-nitrogen content explained an additional 7.3%-14.2% of the variance in rooting ability, indicating that compared with nitrate-nitrogen content, soluble sugar content of rice seedlings before rooting was more dominant in affecting rooting ability. This study provides direct evidence of the relationship between the rooting ability and endogenous soluble sugar content of rice seedlings.