Rodents previously (Trial 1) experienced in the elevated plus-maze (EPM) apparatus no longer respond to anxiolytic-like drugs during retesting (Trial 2). In view of the fact that the dorsolateral periaqueductal gray (dlPAG) modulates fear/anxiety-like behavior, the present study sought to determine its role in this phenomenon. In order to address this issue, EPM-experienced rats that had received lidocaine, a drug which produces a reversible functional deactivation, intra-dlPAG pre-Trial 1, post-Trial 1 or pre-Trial 2, were systemically injected with the benzodiazepine midazolam and submitted to the EPM apparatus. According to the results, 0.25 mg/kg midazolam increased open arms exploration and reduced risk assessment behavior, suggesting an anxiolytic-like effect in EPM-naive rats, regardless of the intra-dlPAG treatment. EPM-experienced rats administered with midazolam only displayed a similar pattern of behavior when lidocaine was administered intra-dlPAG pre-Trial 2, but not pre- or post-Trial 1. These effects were observed in the absence of changes in enclosed arms entries, an EPM general exploratory activity index. The present results suggest that an increased activity of the dlPAG during Trial 2 would explain the lack of anxiolytic-like effect of drugs elicited by prior EPM test experience.