Following the association of a neutral stimulus (conditioned stimulus, CS) with a biologically significant stimulus (unconditioned stimulus, US), CS-alone presentations generate extinction: a decline in the conditioned response. Many studies have revealed the neural substrates of fear extinction; however, a few have identified the brain regions responsible for appetitive extinction. Midbrain dopamine neurons are activated by presenting a reward or predictable reward cue, whereas the cue signaling the absence of reward activates the lateral habenula (LHb) neurons. We examined the engagement of the LHb in appetitive extinction. In the first phase, rats first received pairings of a CS (light) with US delivery (food pellets). In the second phase, rats in the CS-alone group underwent four CS-alone presentations, whereas those in the paired group received four pairings of light with food pellets. We also included a comparison group for CS-alone presentations: rats were placed in the training box without CS or US exposures in the first phase and received four CS-alone presentations in the second phase. Thirty minutes after the second phase, c-Fos levels in the ventral tegmental area (VTA), substantia nigra pars compacta (SNc), and LHb in these groups were measured. c-Fos levels in the LHb were higher in the paired-CS-alone group than in the paired-paired and comparison groups, while those in the VTA and SNc were significantly higher in the paired-paired group than in the other groups. On examination of LHb neurotoxic lesion effects on the decline of conditioned food-cup responses when a CS was repeatedly presented with no US, LHb lesions decelerated the decline in conditioned food-cup responses, suggesting a crucial role of LHb in appetitive extinction.