Early-life neglect in critical developmental periods has been associated with emotional and cognitive consequences. Maternal separation (MS) has been commonly used as a rodent model to identify the developmental effects of child neglect. However, reports have shown considerable variability in behavioral results from MS studies in both mice and rats. Difficulties in developing reliable child neglect models have impeded advances in identifying the effects of early-life stress. Accumulating evidence shows that neuronal intrinsic excitability plays an important role in information processing and storage in the brain. The prefrontal cortex (PFC) integrates information from many cortical and subcortical structures. No studies to date have examined the impact of early-life stress on glutamatergic neuronal excitability in the PFC. This study aimed to develop a reliable child neglect rat model and observe glutamatergic neuronal excitability in the PFC. An MS with early weaning (MSEW) rat model was developed. Rats were separated from the dam for 4 h per day on postnatal days (PNDs) 2–5 and for 8 h per day on PNDs 6–16 and then weaned on PND 17. A battery of behavioral tests was used to assess anxiety-like behavior, coping behavior, working memory, spatial reference memory, and fear memory. The action potentials (APs) of glutamatergic neuronal membranes were recorded. MSEW resulted in anxiety-like behavior, a passive coping strategy and increased fear memory in male rats and decreased locomotor activity in both sexes. MSEW slightly impaired working memory during non-stressful situations in female rats but did not change spatial reference memory or associative learning under stressful circumstances in either sex. MSEW reduced the number of glutamatergic neuron APs in male rats. Our findings showed that MS with early weaning induced anxiety-like behavior in male rats. The reduced glutamatergic neuronal excitability may be associated with the emotional alteration induced by MSEW in male rats. In addition, MSEW induced adaptive modification, which depended on a non-stressful context.