Intolerance of uncertainty (IU), or the increased affective response to situations with uncertain outcomes, is an important component process of anxiety disorders. Increased IU is observed in panic disorder (PD), obsessive compulsive disorder (OCD) and generalized anxiety disorder (GAD), and is thought to relate to dysfunctional behaviors and thought patterns in these disorders. Identifying what brain systems are associated with IU would contribute to a comprehensive model of anxiety processing, and increase our understanding of the neurobiology of anxiety disorders. Here, we used a behavioral task, Wall of Faces (WOFs), during functional magnetic resonance imaging (fMRI), which probes both affect and ambiguity, to examine the neural circuitry of IU in 14 (10 females) college age (18.8 years) subjects. All subjects completed the Intolerance of Uncertainty Scale (IUS), Anxiety Sensitivity Index (ASI), and a measure of neuroticism (i.e. the NEO-N). IUS scores but neither ASI nor NEO-N scores, correlated positively with activation in bilateral insula during affective ambiguity. Thus, the experience of IU during certain types of emotion processing may relate to the degree to which bilateral insula processes uncertainty. Previously observed insula hyperactivity in anxiety disorder individuals may therefore be directly linked to altered processes of uncertainty.