Despite the increasing use of virtual reality, the impact on cerebral representation of topographical knowledge of learning by virtual reality rather than by actual locomotion has never been investigated. To tackle this challenging issue, we conducted an experiment wherein participants learned an immersive virtual environment using a joystick. The following day, participants' brain activity was monitored by functional magnetic resonance imaging while they mentally estimated distances in this environment. Results were compared with that of participants performing the same task but having learned the real version of the environment by actual walking. We detected a large set of areas shared by both groups including the parieto-frontal areas and the parahippocampal gyrus. More importantly, although participants of both groups performed the same mental task and exhibited similar behavioral performances, they differed at the brain activity level. Unlike real learners, virtual learners activated a left-lateralized network associated with tool manipulation and action semantics. This demonstrated that a neural fingerprint distinguishing virtual from real learning persists when subjects use a mental representation of the learnt environment with equivalent performances. Hum Brain Mapp, 2010. © 2009 Wiley-Liss, Inc.