Cotton stalk is one of the most high-quality biomass resources with a wide range of applications, such as building boards, chemical product raw materials, fuel, and papermaking. China is one of the major cotton-producing countries in the world. The planting area (3×106 hm²) has been ranked third in 2022, leading to the very abundant cotton stalk resources. The cotton stalks can be fully utilized to accelerate the green and low-carbon development of agriculture. Mechanical harvesting can be expected to efficiently remove the cotton stalks from the field. However, the current uprooting machine also needs to be improved and optimized for the cotton stalk. In this research, a clamping-type uprooting device with variable stiffness was designed for a high uprooting rate and low clamping breakage rate. The variable rigidity clamping was adjusted as follows. The flexible rubber clamping was used to prevent the cotton stalk from breaking; A rigid support structure was designed on the outside of the rubber block to obtain a greater clamping force. The better performance was achieved by clamping tightly without breaking the cotton stalk. The uprooting was also improved for the two scenarios of breakage and slippage. The device often consisted of a dividing disk, variable stiffness clamping and pulling mechanism, and tensioning guide mechanism. The dividing disk was used for the orderly feeding of cotton stalks; The variable stiffness clamping and pulling mechanism was used to realize the clamping and pulling of cotton stalks; The tensioning guide mechanism was to effectively control the clamping force and gap of cotton stalks. Two sets of chains were utilized to drive multiple clamping blocks, indicating a simple and reliable structure. Moreover, the frictional heating of traditional flexible belt mechanisms was avoided in this structure. A mechanical analysis was carried out on the cotton stalk pulling motion and the interaction between the variable stiffness clamp and the cotton stalk. The critical structural dimensions and operating parameter ranges were determined for the variable stiffness clamping device. A systematic investigation was made to clarify the influencing factors on the quality of clamping and pulling. The response surface method (RSM) was used to analyze the effects of the forward speed, the rotational speed of the active sprocket, and the clamping force on the stalk uprooting performance of the variable stiffness clamping and pulling device. The field test validated the simulation. The results showed that there was very consistency between the experimental and theoretical predictions, when the forward speed was 0.68 m/s, and the sprocket speed was 95 r/min. The tension force was 1 792 N, and the cotton stalk uprooting rate was 94.70%. The relative error between the measured and predicted uprooting rate value was 1.67%, which was less than 5%. The leakage rate and breakage rate of variable stiffness clamping and uprooting devices were 3.99% and 1.32%, respectively, compared with the existing devices of 5.19% and 3.68%, respectively. Therefore, the leakage rate and breakage rate of the device were reduced by 23.1% and 64.13%, respectively, compared with the former. The variable stiffness gripper effectively reduced the cotton stalk breakage to realize the whole stalk uprooting. The finding can provide new ideas to design the variable stiffness clamps, in order to optimize and improve the cotton stalk pullers.