This study aims to propose a reliability-based framework for analyzing the effect of truck platooning on bridge substructures. To achieve this purpose, the dynamic truck platooning–bridge interaction model was developed to calculate the dynamic load allowance (DLA) under varying truck platooning configurations and road conditions. The probabilistic model for DLAs was established based on numerical results. Meanwhile, the finite element model for the bridge pile shaft, considering the uncertainty of surrounding soil, was created for obtaining the probabilistic load capacity model. Then, Monte Carlo sampling method was employed to calculate the pile shaft reliability index using the developed load demand and capacity models. The effects of truck velocity, inter-truck spacing, number of trucks, and road conditions on the pile shaft performance at serviceability limit state were discussed. The results showed that the pile shaft performance could be negatively affected due to the dynamic impact of truck platooning. The number of trucks and inter-truck spacing should be limited to reduce the axial loads sustained by the pile shaft. Poor road conditions could significantly increase the dynamic response of bridges under truck platooning impacts, thus leading to considerable reduction in the pile shaft reliability.