The dynamic model of high-speed trains (HSTs) is nonlinear and uncertain; hence, with the decrease in the running interval of HSTs, an accurate and safe train operation control algorithm is required. In this study, an adaptive output feedback trajectory tracking control method for HSTs is proposed on the basis of neural network observers. The proposed method aims to solve problems, such as the immeasurable speed, model parameter disturbance, and unknown external disturbance of HSTs. In this method, a neural network adaptive observer is designed to estimate the velocity of an HST. Another neural network model is used to approximate the model uncertainties. Moreover, a robust controller is constructed by considering the train position and velocity tracking errors. In the proposed observer/controller, the bound function of estimator errors is introduced to increase the accuracy and safety of the tracking system. Furthermore, the adaptive update value of the neural networks, output weights, and bound function are performed online. All adaptive algorithms and the observer/controller are synthesized in nonlinear control systems. The error signals of the closed-loop trajectory tracking system are uniformly and eventually bounded through a formal proof on the basis of the Lyapunov methods. Simulation examples illustrate that the proposed controller is robust and has excellent tracking accuracy for system model parameter and external disturbance.