Background: Lumbar puncture (LP) is a commonly performed medical procedure in a wide range of indications. Virtual reality (VR) provides a stimulating, safe and efficient learning environment. We report the design and the evaluation of a three dimensions (3D) video for LP training. Methods: We recorded a stereoscopic 180-degrees 3D video from two LPs performed in clinical settings in Fernand Widal Lariboisière University Hospital, Paris, France. The video was administered to third-year medical students as well as to a residents and attendings group during LP simulation-based training sessions. Results: On 168 participants (108 novice third-year medical students, and 60 residents and attendings with prior LP experience), satisfaction after video exposure was high (rated 4.7 ± 0.6 on a 5-point scale). No significant discomfort was reported (comfort score graded 4.5 ± 0.8 on 5). LP-naive students displayed higher satisfaction and perceived benefit than users with prior LP experience (overall, P < 0.05). Trainees evaluated favorably the 3D feature and supported the development of similar tutorials for other medical procedures (respectively, 3.9 ± 1.1 and 4.4 ± 0.9 on 5). Conclusion: We report our experience with a 3D video for LP training. VR support could increase knowledge retention and skill acquisition in association to LP simulation training.