In order to realize the rapid start-up of vehicle-carried inertial navigation system, the paper studies the in-motion alignment method based on onboard rotating inertial navigation system (RINS) and GPS velocity information. According to the rotation characteristics of the rotating inertial navigation system, the Kalman filter model was established, including the installation angles between gyros, the lever arm and the asynchronous time, by measuring the velocity difference between RINS and GPS. Besides, multiple simulations and experimental verifications of the proposed in-motion alignment method have been carried out. The results show that the proposed in-motion alignment method can realize the rapid estimation of platform deflection angle, gyro installation errors, lever arm and asynchronous time of RINS in the process of motion, which verifies the effectiveness of the proposed in-motion alignment method.