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Fruit nondestructive picking is one of the key technologies of developing harvesting robot. A nondestructive picking end-effector of kiwifruit was studied. Firstly, based on the artificial way of kiwifruit picking and the biology characteristics of kiwifruit stem, a fruit picking method for robot was proposed, which need to separate the fruit from stem and hold the fruit to prevent it dropping. Then, the picking method was verified by a special designed separation test of fruit and its stem. After that, an end-effector was designed and manufactured based on the fruit picking method, which approached a fruit from the bottom, and enveloped and grabbed the fruit from two sides, and then rotated up to separate the fruit from stem. In the end, the performance of end effector prototype was tested on the most common cultivar ‘Hayward’ at the Meixian Kiwifruit Experimental Station of Northwest A&F University. The results showed that the proposed picking method could separate the fruit successfully with the least force of 1.3N when the angle between fruit and stem was set as 60°, which is not significantly different from the manual picking of which the angle between fruit and stem is approximately to 90° in normal. The end effector was tested on 68 samples (28 in the morning, 25 in the noon, and 15 in the night). All of them were successfully picked and held by the end-effector. Among them, two samples were picked with stem which might be caused by the reason of that the fruit is not ripe enough to be harvested. All the picked fruit were free of damage until ripen for eating. In all, the end effector could effectively solve the problems of separating the adjacent fruits, grab a single fruit with an accuracy of 100%, and pick and hold it nondestructively. The success rate of picking was 96.0% and the average picking time was 22s.