Anxiety is one of the most common mental disorders. Neurotensin (NT) is a neuropeptide widely distributed in the central nervous system, involved in the pathophysiology of many neural and psychiatric disorders such as anxiety. However, the neural substrates mediating NT's effect on the regulation of anxiety have not been fully identified. The medial septum (MS) is a crucial brain region in regulating anxiety and expresses neurotensin receptor 1 (NTS1). In the current study, we examined the role of NT/NTS1 in the MS on the anxiety-like behaviors of rats in non-stress and acute stress conditions. We reported that intra-MS infusion of NT produced remarkable anxiogenic effect in behavioral tests. The anxiogenic effect could be blocked by NTS1 antagonist SR48692 pretreatment. Microinjection of NTS1 antagonist to block endogenously released NT in the MS had no effect on anxiety-like behaviors in non-stressed rats, but significantly reduced anxiety in acute restraint stressed rats. Moreover, molecular knockdown of NTS1 in the MS ameliorated anxiety induced by acute restraint stress, also confirming the pharmacological results. Our study implicates NT and its receptors as potential targets for therapeutic interventions of psychiatric diseases, such as anxiety.