<p>Ventral tegmental area (VTA) is typically involved in heroin reward, but it is still unclear whether the VTA or neural projections containing VTA are also involved in conditioned cues (CS)-induced heroin-seeking behavior. In present study, rats were first trained to self-administer heroin for 14 days, then undergone withdrawal for another 14 days. Subsequently, part of rats were assessed CS-induced heroin-seeking behaviors and tested c-Fos protein expression in brain areas after microinjection with saline, neostigmine or tetrodotoxin (TTX) into the VTA. Part of rats were tested CS-induced heroin-seeking behaviors after chemogenetic inhibition of the dopaminergic projection from VTA to paraventricular thalamus (PVT) by stimulating with clozapine N-oxide (CNO). We found that CS-induced heroin-seeking behaviors could be enhanced by microinjection with neostigmine, but weakened by microinjection with TTX, into the VTA. The expression of c-Fos immunoreactive neurons were increased in the PVT and nucleus accumbens (NAc) after neostigmine treatment, but decreased in the PVT, NAc and lateral habenula (LHb) after TTX treatment. Chemogenetic inhibition of the dopaminergic projection from VTA to PVT could significantly inhibit CS-induced heroin-seeking behaviors of rats. Our results suggest that stimulating VTA promotes CS-induced heroin-seeking behavior, and the underlying neural circuits may involve the projection from VTA to PVT. </p>