Chemotherapy agents are cytotoxic materials. Thus, there is a need for the operators to be familiar with the knowledge and procedures before operation. We conducted a randomized controlled trial investigating the effectiveness of an immersive 3D VR teaching of chemotherapy administration operated in a smartphone coupled with a visual and audio device. We adopted a two-arm single-blind design and recruited 83 nurses, and they were randomized using a cluster approach. The VR group learned chemotherapy administration through VR, while the controlled group learned through document reading. The Knowledge and Attitude of Chemotherapy Administration (KACA) was administrated before the intervention, while the Objective Structured Clinical Examination (OSCE) and the Checklist of Action Accomplishment (CAA) were administrated one month after the intervention. The VR group scored higher than the controlled group in the CAA (95.69 ± 5.37 vs. 91.98 ± 9.31, p = 0.02) and the OSCE (73.07 ± 10.99 vs. 67.44 ±10.65, p = 0.02). Stepwise regression demonstrated that service years, an education level of undergraduate or above, and VR exposure contributed positively to the OSCE score (adjusted R2 = 0.194, p = 0.028). The use of VR improves the learning efficacy of chemotherapy administration in non-oncology nurses. We recommend using VR as a teaching tool for chemotherapy administration and other chemotherapy-related skills in a VR learning group with senior nurses with higher education levels as advisors. The study provides an approach to online training, especially during the COVID-19 pandemic. (CONSORT 2010 guidelines, registry number: NCT 04840732).