Abstract: This study aims to observe and simulate the internal movement of fungal material substrate during the piercing and laying of black fungal sticks using the discrete element method (DEM). The contact parameters of fungi were calibrated on the different sizes within the cultivation sticks. Key parameters, such as particle stiffness, friction coefficients, and damping factors, were adjusted to replicate the interactions among fungal particles. DEM simulations were used to accurately reflect real-world mechanical behavior. Both physical experiments and DEM simulations were conducted on piercing after calibration. The physical experiments were conducted to measure the key substrate parameters, particularly for the influences of repose angle on the substrate settles. The DEM simulations were fine-tuned to closely match the real-life scenarios after measurements. In simulations, virtual tools were inserted into the modeled fungal sticks, in order to observe substrate behavior under controlled conditions. The comparison was performed on the depth and diameter of the holes created during piercing, in order to validate the accuracy of the DEM model. The simulation results were compared with experimental data obtained from a fully automatic piercing machine. The relative errors between the simulation and experimental data were 3.6% for the hole depth and 4.5% for the hole diameter. The low error margins confirmed that the high accuracy of the model was achieved to replicate the real-world conditions, providing for the reliability of the DEM model in the subsequent simulations. In addition to the piercing simulations, a systematic investigation was also made to explore the impact of landing shock on the deformation of fruiting holes within the fungal sticks. The falling behavior of fungal sticks was simulated in a semi-automatic laying machine. Displacement velocities of substrate particles around the ear holes were measured at three positions along the fungal stick: the top, middle, and bottom. The velocities were recorded as 441, 621, and 1 115 mm/s, respectively. There was the most significant deformation at the bottom of the stick and then decreased towards the top. The diameter and depth of the bottom ear holes were 12.3% and 14.3% smaller, respectively, compared with the top ones. The minimum aperture diameter and depth were 3.2 and 32.5 mm after simulation, respectively, fully meeting the requirements for ear production. Therefore, the fungal sticks remained suitable for agricultural use, even after deformation under landing shock. The semi-automatic laying machine was effective with the acceptable deformation for practical agricultural production. In conclusion, the DEM model was validated to accurately simulate the piercing and laying in the internal movement of fungal material within black fungal sticks. The insights were gained on the deformation caused by landing shock. The findings can greatly contribute to the design and operation of machinery in mushroom cultivation. The quality and functionality of fungal sticks were maintained throughout the production. The DEM model can be expected to improve agricultural practices in mushroom cultivation.