We aimed to determine the short-term effects of early-life stress in the form of maternal separation (MS) on anxiety-like behavior in male rat pups. In order to assess anxiety, we measured 40 kHz separation-induced ultrasonic vocalizations (USV) on postnatal day (PND) 11. We further aimed to evaluate the potential involvement of two neurochemical systems known to regulate social and anxiety-like behaviors throughout life: oxytocin (OT) and fibroblast growth factor 2 (FGF2). For these purposes, we tested the effects of neonatal administration (on PND1) of an acute dose of FGF2 on USV and its potential interaction with MS. In addition, we validated the anxiolytic effects of OT and measured oxytocin receptor (OTR) gene expression, binding and epigenetic regulation via histone acetylation. Our results show that MS potentiated USV while acute administration of OT and FGF2 attenuated them. Further, we found that both FGF2 and MS increased OTR gene expression and the association of acH3K14 with the OTR promoter in the bed nucleus of the stria terminalis (BNST). Comparable changes, though not as pronounced, were also found for the central amygdala (CeA). Our findings suggest that FGF2 may exert its anxiolytic effects in male MS rats by a compensatory increase in the acetylation of the OTR promoter to overcome reduced OT levels in the BNST.