This paper addresses the generation mechanism and avoidance method of negative stiffness during in-Hand manipulation with underactuated compliant hands. Firstly, a planar hand with two three-jointed fingers manipulating a rectangular is set, and a quasi-static underactuated operation model is established. Secondly, based on this simulation model, we investigated the stiffness evolution during in-hand manipulation, and analyze the influence factors of system stiffness. Finally, a stiffness regulation method is developed to avoid negative stiffness during in-hand manipulation. The method is validated by simulation. The research results are beneficial to improve the performance of underactuated in-hand manipulation.