Background: Virtual reality (VR) technologies have been developed to assist education and training. Although recent research suggested that the application of VR led to effective learning and training outcomes, investigations concerning the acceptance of these VR systems are needed to better urge learners and trainees to be active adopters. Objective: This study aimed to create a theoretical model to examine how determining factors from relevant theories of technology acceptance can be used to explain the acceptance of a novel VR-assisted mental rotation (MR) training system created by our research team to better understand how to encourage learners to use VR technology to enhance their spatial ability. Methods: Stereo and interactive MR tasks based on Shepard and Metzler’s pencil and paper test for MR ability were created. The participants completed a set of MR tasks using 3D glasses and stereoscopic display and a 6-degree-of-freedom joystick controller. Following task completion, psychometric constructs from theories and previous studies (ie, perceived ease of use, perceived enjoyment, attitude, satisfaction, and behavioral intention to use the system) were used to measure relevant factors influencing behavior intentions. Results: The statistical technique of partial least squares structural equation modeling was applied to analyze the data. The model explained 47.7% of the novel, VR-assisted MR training system’s adoption intention, which suggests that the model has moderate explanatory power. Direct and indirect effects were also interpreted. Conclusions: The findings of this study have both theoretical and practical importance not only for MR training but also for other VR-assisted education. The results can extend current theories from the context of information systems to educational and training technology, specifically for the use of VR-assisted systems and devices. The empirical evidence has practical implications for educators, technology developers, and policy makers regarding MR training.