Objective This study aims to evaluate the effect of in-vehicle audio warning at flashing-light-controlled grade crossings based on driving simulation and eye-tracking systems. Background Collisions at flashing-light-controlled grade crossings have severe consequences. In-vehicle audio warning has the potential to regulate driver behavior. However, whether this improvement occurs through priming drivers’ visual search patterns is not yet clear. Method Drivers’ visual activity and behaviors were recorded. The effect of a warning was tested with a series of flashing light trigger times (FLTTs) ranging from 2s to 6s with a 1s increment. Different driving conditions (i.e., clear and fog) and driver experience were considered in the experiment design. Results Warnings could guide the allocation of both overt and covert attention, as well as raise drivers’ situation awareness, manifesting as the enhanced perception of signs and better understanding of the flashing red light. Significant improvement in the stop-compliance rate was found in warning scenarios, particularly with a late FLTT. The decreased saccade duration and increased fixation duration on the signal implied a dilemma-zone effect when the FLTT was lower than 4s. Furthermore, reduced fixation duration on signs and signals was found in foggy conditions. Non-professional drivers had a wider search range than their counterparts. Conclusion In-vehicle audio warning is an effective countermeasure for improving crossing safety by optimizing visual search strategy. Application In-vehicle audio warnings warrant promotion at grade crossings based on the driver assistance system.