Abstract Background/Purpose: In previous studies using mucosal scrapings or whole-bowel homogenates, epidermal growth factor (EGF) augments adaptation after massive small bowel resection (SBR). The purpose of this study was to determine directly the effect of adaptation and EGF on enterocyte differentiation using an explicit enterocyte cell population. Methods: Male ICR mice underwent 50% proximal SBR or sham (bowel transection-reanastomosis) and were selected randomly to either orogastric saline or EGF (50 μg/kg/d). After 3 days, enterocytes were isolated from the remnant ileum by mechanical vibration and assayed for DNA and protein content as well as sucrase and alkaline phosphatase (AlkP) activity. Results: Ileal wet weight, enterocyte protein, and DNA content were increased significantly after SBR and boosted even further with EGF. When normalized for protein, SBR caused an increase in AlkP and sucrase activity, and EGF treatment caused AlkP and sucrase activity to return to baseline. Conclusions: EGF enhances adaptation; however, when normalized for protein, the activity of two enterocyte-specific enzymes was not significantly altered by EGF. This analysis of an explicit enterocyte population supports the notion that the beneficial effects of EGF are more likely caused by increased numbers of enterocytes rather than an increase in the functional activity of each individual cell.