Objective To investigate the correlation of the serum levels of intestinal fatty acid binding protein (I-FABP) and D-lactate (D-Lac) with ischemic time and intestinal mucosa injury in rats with acute mesenteric ischemia. Methods A total of 60 rats were randomly and equally divided into 6 groups, that is, sham-operation group and 5 mesenteric ischemia groups according to mesenteric artery occluded for 30, 60, 90, 120 and 150 min respectively. Their serum levels of I-FABP and D-Lac were determined by ELISA. The content of I-FABP in the damaged intestinal mucosa was measured by immunohistochemical assay. Histopathological analysis was performed in the injured intestinal mucosa to evaluate the score of injury. The correlation of serum levels of I-FABP and D-Lac with tissue injury was analyzed. Results The serum I-FABP level was significantly increased from 30 min after ischemia, and reached a peak at 90 min [(1741.37±184.12)mg/L], and the cells positive to I-FABP accounted for 72.5% (P＜0.05). The serum level of D-Lac was significantly increased from 60 min after ischemia and showed a time-dependent manner (P＜0.05). The score of injury was also increased with the extent of ischemic time. Spearman’s rank correlation non-parameter test indicated that the serum level of D-Lac was positively correlated with intestinal mucosa injury (r＝1), but that of I-FABP had no such correlation (r＝0.6). Conclusions Serum I-FABP is increased at the early stage of acute mesenteric ischemia, and then after reaching peak, decreased with the elapse of ischemic time. Serum D-Lac is obviously increased at the late stage, and might be used to predict the severity of intestinal ischemic injury.