Abstract: Abstract：Separating seedling is highly lacking to implement the planting operation in mechanized transplanting of rapeseed substrate block seedlings. In this study, a novel separating seedling device with a pair of rollers was designed in a transplanter, particularly for the rapeseed substrate block seedlings using a degree of freedom (DOF) constraint of a mechanical system. Two stages were divided in the working process of the device, including pneumatic seedling separation and counter-roller seedling collection. As such, the combination of pneumatic seedling separation and counter-roller wheel was used to realize the stable and synchronous seedling separation of each row. Kinematic models were also constructed for the separate seedling and picking stage in the process of transplanting. Some key parameters were optimized as follows: the linear speed of the synchronous belt ranged from 66.67 to 133.33 mm/s, while the roller speed ranged from 192.46 to 294.67 mm/s, and the angle of the seedling platform was 30° to 45°. A quadratic rotation orthogonal experiment was carried out to explore the effects of the linear speed of synchronous belt and roller, as well as installation angle of seedling loading platform on the success and synchronization rate in seedling picking. The results showed that the performance of picking seedlings was better than before, when the speed of the synchronous belt was 120.9 mm/s, the linear speed of the roller was 238.2 mm/s, and the angle of the seedling platform was 31.9°. The success rate and synchronization rate were also 93.42% and 94.42%, respectively. It was found that with the increase of the angle of the seedling platform in the bench test, the conveying fluency of seedling substrate block decreased significantly, together with some toppling of seedling, where the seedling substrate block moved from the synchronous belt to the seedling platform. The reason was that the stability of the seedling dropped significantly, as the angle of the seedling platform increased, thereby easy to topple, where there was an obvious decrease in the distance between the seedling substrate block and the bottom of the ditch. Correspondingly, the seedling was easy to cause the collapse, whether the linear speed of the synchronous belt was too high or low. The error was accumulated constantly, as the number of seedlings increased, further leading to the missing planting and heavy load. Once the horizontal speed of the roller was consistent with the forward speed of a machine, the speed of the seedling dropping was achieved at zero, while the stability of the seedling was much better. But when the speed changed greatly, the stability of the seedling picking using a counter roller was getting worse, while the seedling dropping was easy to occur dumping. In any way, a field experiment demonstrated that the success rate of collection seedlings was 88.69%, and the synchronous rate of collection seedlings was 92.72%, meeting the technical requirements of separating seedlings of rapeseed substrate block seedlings transplanter. The finding can provide a sound reference to the structural improvement of separating seedlings device in the rapeseed substrate block seedlings transplanter for modern mechanized agriculture.