Abstract With increase in computer usage, three-dimensional (3D) virtual environments (VEs) have become new areas for navigation and spatial learning. Second Life (SL) has become one of the most popular 3D VEs that is used as a learning environment. This study focuses on the issue of spatial learning during virtual navigation in a multi-level desktop VE designed in SL. The usability of SL as a tool for spatial learning in interior architectural design is investigated by analyzing virtual navigation paths, gender differences and sense of presence. The study is conducted with 90 interior architecture students studying at Bilkent University, Ankara, Turkey. Results indicated that SL can be an effective tool for individuals to learn an environment and for interior architects to improve their designs by learning from the navigation behaviors of the users.