Although pancreatic β-cells are capable of adapting their mass in response to insulin requirements, evidence has shown that a dietary insult could compromise this ability. Fetal malnutrition has been linked to low birth weight and the development of type 2 diabetes later in life, while reduced β-cell mass has been reported in adult rats fed a high-fat diet (HFD). Reported here are the effects of exposure to a HFD, during different periods of gestation, on neonatal rat weight and β- and α-cell development. The experimental groups were composed of neonatal offspring obtained from Wistar rats fed a high-fat (40% as energy) diet for either the first (HF1), second (HF2), or third (HF3) week, or all three (HF1–3) weeks of gestation. Neonatal weights and circulating glucose and insulin concentrations were measured on postnatal day 1, after which the pancreata were excised and processed for histological immunocytochemical examination and image analysis. HF1 and HF2 neonates were hypoglycemic, whereas HF1–3 neonates were hyperglycemic. Low birth weights were observed only in HF1 neonates. No significant differences were detected in the circulating insulin concentrations in the neonates, although β-cell volume and numbers were reduced in HF1–3 neonates. β-cell numbers also declined in HF1 and HF3 neonates. α-cell volume, number and size were, however, increased in HF1–3 neonates. α-cell size was also increased in HF1 and HF3 neonates. In neonates, exposure to a maternal HFD throughout gestation was found to have the most adverse effect on β-cell development and resulted in hyperglycemia.