Although gut lipid absorptive capacity is depressed after trauma and hemorrhagic shock, it remains unknown whether this is associated with an impairment in the enterocyte triglyceride (TG) synthesis, and if so, whether villus or crypt cells participate in the lipid absorption under such conditions. To study this, midline laparotomy (i.e., trauma induction) was performed in the rats, and then the animals were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of shed blood volume was returned in the form of Ringer's lactate. The rats were then resuscitated with four times the volume of maximal bleedout with Ringer's lactate over 60 min. For lipid loading test, 1 mL of olive oil was given intraduodenally as a bolus injection upon the completion of resuscitation. The enterocytes along the crypt-villus axis from the proximal small intestine were isolated at either 0, 1.5, or 5 h after fat feeding. The intracellular and pre-chylomicron TG contents as well as the microsomal enzyme activities associated with TG synthesis were determined thereafter. The results indicated that intracellular and pre-chylomicron TG contents and lipid esterifying enzyme (i.e., acyl-coenzyme A:monoglyceride acyltransferase) activity decreased significantly in villus tip cells after trauma-hemorrhage and resuscitation. The intracellular TG content and lipid esterifying enzyme activity in crypt cells after trauma and hemorrhagic shock were also reduced. However, there was no significant decrease in pre-chylomicron TG content in the crypt cells isolated from trauma-hemorrhaged rats. It is unlikely that crypt cells participate in lipid transport after trauma and hemorrhagic shock. In contrast, the TG synthesis in villus tip cells decreased significantly following trauma-hemorrhage and resuscitation, which may contribute, at least in part, to the impaired gut lipid absorptive capacity observed under such conditions.