Inhalation of low concentrations of carbon dioxide (CO2) triggers anxious behaviours in rodents via chemosensors in the amygdala, and increases anxiety, autonomic arousal and hypervigilance in healthy humans. However, it is not known whether CO2 inhalation modulates defensive behaviours coordinated by this network in humans. We examined the effect of 7.5% CO2 challenge on the defensive eye-blink startle response. A total of 27 healthy volunteers completed an affective startle task during inhalation of 7.5% CO2 and air. The magnitude and latency of startle eye-blinks were recorded whilst participants viewed aversive and neutral pictures. We found that 7.5% CO2 increased state anxiety and raised concurrent measures of skin conductance and heart rate (HR). CO2 challenge did not increase startle magnitude, but slowed the onset of startle eye-blinks. The effect of CO2 challenge on HR covaried with its effects on both subjective anxiety and startle latency. Our findings are discussed with reference to startle profiles during conditions of interoceptive threat, increased cognitive load and in populations characterised by anxiety, compared with acute fear and panic.