Background It is unknown how traumatic brain injury (TBI) increases risk for posttraumatic stress disorder (PTSD). One potential mechanism is via alteration of fear‐learning processes that could affect responses to trauma memories and cues. We utilized a prospective, longitudinal design to determine if TBI is associated with altered fear learning and extinction, and if fear processing mediates effects of TBI on PTSD symptom change. Methods Eight hundred fifty two active‐duty Marines and Navy Corpsmen were assessed before and after deployment. Assessments included TBI history, PTSD symptoms, combat trauma and deployment stress, and a fear‐potentiated startle task of fear acquisition and extinction. Startle response and self‐reported expectancy and anxiety served as measures of fear conditioning, and PTSD symptoms were measured with the Clinician‐Administered PTSD Scale. Results Individuals endorsing “multiple hit” exposure (both deployment TBI and a prior TBI) showed the strongest fear acquisition and highest fear expression compared to groups without multiple hits. Extinction did not differ across groups. Endorsing a deployment TBI was associated with higher anxiety to the fear cue compared to those without deployment TBI. The association of deployment TBI with increased postdeployment PTSD symptoms was mediated by postdeployment fear expression when recent prior‐TBI exposure was included as a moderator. TBI associations with increased response to threat cues and PTSD symptoms remained when controlling for deployment trauma and postdeployment PTSD diagnosis. Conclusions Deployment TBI, and multiple‐hit TBI in particular, are associated with increases in conditioned fear learning and expression that may contribute to risk for developing PTSD symptoms.