Cue reactivity is an established procedure in addictions research for examining the subjective experience and neural basis of craving. This experiment sought to quantify cue-related brain responses in gambling disorder using personally tailored cues in conjunction with subjective craving, as well as a comparison with appetitive non-gambling stimuli. Participants with gambling disorder (n=19) attending treatment and 19 controls viewed personally tailored blocks of gambling-related cues, as well as neutral cues and highly appetitive (food) images during a functional magnetic resonance imaging (fMRI) scan performed ~2–3 h after a usual meal. fMRI analysis examined cue-related brain activity, cue-related changes in connectivity and associations with block-by-block craving ratings. Craving ratings in the participants with gambling disorder increased following gambling cues compared with non-gambling cues. fMRI analysis revealed group differences in left insula and anterior cingulate cortex, with the gambling disorder group showing greater reactivity to the gambling cues, but no differences to the food cues. In participants with gambling disorder, craving to gamble correlated positively with gambling cue-related activity in the bilateral insula and ventral striatum, and negatively with functional connectivity between the ventral striatum and the medial prefrontal cortex. Gambling cues, but not food cues, elicit increased brain responses in reward-related circuitry in individuals with gambling disorder (compared with controls), providing support for the incentive sensitization theory of addiction. Activity in the insula co-varied with craving intensity, and may be a target for interventions.