Fear of the unknown is thought to underpin individual differences in self-reported intolerance of uncertainty (IU). Over the last decade, IU has gained substantial interest and now sits at the forefront of anxiety and stress research. Despite the emergence of IU as a construct, there is a dearth of literature on the extent to which the known vs. the unknown during threatening contexts induces fear and anxiety in individuals with high IU. To address this question, we manipulated the known and unknown during a classic anxiety task (n = 93). Throughout the task, we measured a variety of physiological indices (i.e. startle blink and facial frowning). Higher IU, relative to lower IU, was associated with physiological markers of fear and anxiety (i.e startle blink and facial frowning) during conditions with known, compared to unknown threat. These findings provide evidence that IU-related fear and anxiety during threatening contexts is driven by the perception that something known isn’t correct or complete, rather than when something is entirely unknown. Ultimately, this research will inform future models of IU in relation to anxiety and stress disorders.