Intrinsic functional connectivity detected by functional MRI (fMRI) provides a useful but indirect approach to study the organization of human brain systems. An unresolved question is whether functional connectivity measured by resting-state fMRI reflects anatomical connections. In this study, we used the well-characterized anatomy of cerebrocerebellar circuits to directly test whether intrinsic functional connectivity is associated with an anatomic pathway. Eleven first-episode stroke patients were scanned five times during a period of 6 months, and 11 healthy control subjects were scanned three times within 1 month. In patients with right pontine strokes, the functional connectivity between the right motor cortex and the left cerebellum was selectively reduced. This connectivity pattern was reversed in patients with left pontine strokes. Although factors beyond anatomical connectivity contribute to fMRI measures of functional correlation, these results provide direct evidence that functional connectivity depends on intact connections within a specific polysynaptic pathway.