Diffusion Tensor Imaging (DTI) is a promising new imaging method allowing in vivo mapping of anatomical connections in the living human brain. We combined DTI with functional magnetic resonance imaging (fMRI) to investigate the anatomical relationships between areas involved in visceral sensations in humans. Non-painful and moderately painful rectal distensions were performed in 11 healthy women (38.4+/-3.1years). fMRI was used to analyse the changes in brain activity during both types of distension. Then, DTI was applied for tracking fibers between the main activated regions. Non-painful distension bilaterally activated the PreFrontal Cortex (PFC), the Anterior Cingulate Cortex (ACC) and the right insula. Painful distension bilaterally activated the primary (S1) and secondary (S2) somatosensory cortices, the motor cortex, the frontal inferior gyrus, the thalamus, the insula, the striatum and the cerebellum. DTI revealed direct connections between insula, and the four areas more frequently activated in this study, i.e. ACC, thalamus, S1, S2 and PFC. The combined use of fMRI and DTI in healthy subjects during rectal distension revealed a neural network of visceral sensory perception involving the insula, thalamus, somatosensory cortices, ACC and PFC.