To improve the accuracy of simulation parameters used in discrete element simulation tests for the transplanting operation of the transplanting machine and to facilitate further optimization of crucial components of the transplanting machine, in this paper, the discrete element model of 50-hole plug seedling pots was calibrated and optimized based on the collision impact force between the plug seedling pot and the steel plate measured by a flexible film network tactile pressure sensor. Basic tests determined the contact parameters of the pot, and the initial parameters were screened for significance using the Plackett–Burman test. The pot-steel static friction coefficient, the pot-pot collision restitution coefficient, and the bond radius significantly affected the simulated collision impact force between the pot and the steel plate. According to the relative error value of the impact force between the pot and the steel plate as the evaluation index, the steepest climbing test was carried out on three significant parameters to optimize their value range. Based on the Box–Behnken test, a second-order regression model of the impact force and significant parameters regulating the interaction between the pot and the steel plate was established, where the target impact force between the pot and the steel plate was 11.78 N. The optimal parameter combination is obtained by optimizing the significance parameters: the static friction coefficient between the pot and steel is 0.790, the collision restitution coefficient between the pot and the pot is 0.325, and the bond radius is 1.542 mm. The test results show that the relative error between the actual and simulation tests is only 0.084%. The calibrated parameters of the discrete element model of plug seedling pots are accurate and reliable. The research results presented here can provide a reference for the subsequent transplanting operation simulation of the transplanter.