We investigated the anatomical location of the pedunculopontine nucleus (PPN) in the human brain using diffusion tensor imaging. Forty normal healthy subjects were recruited. To confirm the boundary of the PPN, we analyzed the superior cerebellar peduncle and medial lemniscus using DTI-Studio software. We identified the PPN on red green blue (RGB) images, and defined four points of the PPN and four boundaries of the midbrain: point a – the most anterior point, point b – the most posterior point, point c – the most medial point, point d – the most lateral point; anterior boundary – the line of the most posterior point of the interpeduncular fossa, posterior boundary – the line of the upper part of the inferior colliculus, lateral boundary – the line of the most lateral point of the midbrain, medial boundary – the line of the midline of the midbrain. Points a and b were located at an average of 20.19 and 30.52% from the anterior boundary, respectively. By contrast, points c and d were located at an average of 22.50 and 41.65% from the medial boundary, respectively. We believe that the methodology and data of this study would be helpful in research and procedures on the PPN.