Movement and locomotion are fundamental for goal-directed behaviours in diverse invertebrate and vertebrate organisms. Integrating higher brain centers with evolutionarily conserved descending brainstem locomotor command centers plays a key role in orchestrating context-specific behaviours. The mesencephalic locomotor region (MLR), primarily comprised of the cuneiform nucleus (CnF) and pedunculopontine nucleus (PPN) is one such conserved descending locomotor center in diverse vertebrate species shown to produce locomotion. Excitatory glutamatergic neurons of the CnF have been shown to set both speed and gait for high-speed locomotion implicated in defensive flight when activated. The medial zona incerta (mZI) is an important hub, allowing for rapid responses to sensory inputs. Projections from the mZI to CnF exist, forming a critical inhibitory circuit for the expression of exploratory behaviours. Less is known on the information however evolved around excitatory afferents to the CnF and their functional implications. Using both a retrograde and anterograde viral tracing approach, I identified a glutamatergic circuit between the mZI and CnF. Activation of the mZI-CnF glutamatergic circuit did not provoke an increase in locomotion within an open field. However, an increase in aversive behaviour with less time spent in a conditioned chamber as well as exhibited heightened levels of anxiety with increased grooming and immobility was observed. Mouse grimace scale (MGS) testing did not reveal any significant difference in pain-related facial expressions. Together, these results highlight that glutamatergic afferents from the mZI to CnF elicits aversive behaviour that is independent of locomotion. My thesis provides evidence that activation of this circuit is sex-specific, with females showing significant aversion compared to male mice.