Nowadays, pump-turbine units have to experience multiple start-stops every day to balance the power production and consumption on the grid. During the transient process of turbine start-up, the hydraulic forces applied to the head-cover would change dramatically and induce high-level stresses on the head-cover bolts. As key components of large hydraulic turbine units, the head-cover bolts are subjected to tens of thousands of tonnes of hydraulic excitation force during operation. Special attention should be paid to the design of the head-cover bolts of large hydraulic pump-turbine units because these units have high water heads and high hydraulic excitation forces. Therefore, the safe design of the head-cover bolts is extremely important to maintain the operational safety of the whole unit. This paper investigates the flow-induced stress characteristics of the head-cover bolts during turbine start-up in a large prototype pump-turbine unit. A complete 3D fluid model and a corresponding 3D structural model, including the head-cover bolts of the pump-turbine unit, were created. The fluid–structure coupling method was used to calculate the structural stresses caused by fluid flow during turbine start-up. The pressure files during turbine start-up calculated by the CFD tool were transferred and mapped to the finite element model of the structural components of the pump-turbine unit. Subsequently, the flow-induced stress characteristics of the head-cover bolts were numerically simulated. The simulation results showed that the hydraulic excitation force on the head-cover bolts increased significantly during turbine start-up, and the displacement and the stress distributions of different head-cover bolts were not uniform. The calculation methods and conclusions in this paper can also be applied to evaluate the flow-induced stress characteristics of head-cover bolts for similar hydraulic pump-turbine units.