Neuronal ensembles within the infralimbic cortex (IL) and their projections to the nucleus accumbens (NAc) mediate opiate seeking in well-trained rats. However, it is unclear how early this circuitry is recruited during oxycodone self-administration. Here, we used retrograde labelling (CTb) and immunohistochemistry to identify NAc-projecting neurons in the IL that were activated during initial oxycodone seeking. Next, we sought to determine the role of IL neuronal ensembles in initial oxycodone self-administration. We used the Daun02 procedure in male and female Fos-LacZ rats to chemogenetically inactivate IL Fos-expressing neurons at different time points in oxycodone self-administration training: immediately after meeting criteria for acquisition of behaviour and following nine daily sessions with increasing schedules of reinforcement (FR1, FR2 and FR3) in which rats demonstrated stable oxycodone intake under increasing effort to self-administer. We found that Daun02 infusions attenuated oxycodone seeking at both the initial learning and well-trained time points. These results suggest that IL neuronal ensembles are formed during initial learning of oxycodone self-administration and required for the maintenance and expression of oxycodone seeking.