Objective To assess the efficacy and limitations of enteral nutrition supplemented with probiotics in managing severe pancreatitis， we investigated the dynamic alterations of enteric fatty acid-binding protein （I-FABP）， D-lactic acid （D-Lac）， and citrulline. Methods Between June 2021 and June 2024， a total of 153 cases of severe pancreatitis were enrolled from the Department of Gastroenterology at the First Affiliated Hospital of Soochow University and the Department of Critical Care Medicine at Suzhou Hospital Affiliated with Nanjing Medical University. These cases were divided into two groups： A treatment group receiving probiotics in addition to enteral nutrition （EN）， and a control group without probiotic supplementation. The treatment group received a daily dosage of four capsules containing bifidobacterium triple live bacteria. Intestinal biomarkers were assessed at three time points following EN initiation： before initiation （D0）， on day 3 （D3）， and on day 10 （D10） after initiation. Repeated measures ANOVA was employed to analyze the efficacy of the three intestinal biomarkers， with D10 as the endpoint for evaluation. Subgroup analysis was conducted to explore any potential influence of antibiotic usage on probiotic effects. Various conventional evaluation indicators for pancreatitis， including amylase levels， inflammation markers， relevant scores， post-EN intestinal tolerance， 90-day mortality rates， discharge times， and other related outcomes were compared between the two groups. Results The results of a repeated measurement ANOVA showed that： （1） the main effect of I-FABP did not reach statistical significance （P = 0.076）， whereas the interaction effect was statistically significant （F = 10.691， P = 0.001， partial η² = 0.066）； （2） Neither the main effect of D-Lac nor its interaction effect reached statistical significance （P = 0.761 and P = 0.995， respectively； F < 0.004 for both effects， partial η² < 0.001）； （3） Although the main effect of citrulline did not reach statistical significance （P = 0.161）， its interaction effect was found to be statistically significant with a large effect size （F = 32.437， P < 0.001， partial η² = 0.177）. Endpoint efficacy analysis demonstrated that： （1） The I-FABP treatment group exhibited significantly higher pre-reduction levels compared to the control group （Difference in LS Mean （95% CI） -0.43 （-0.47 ~ -0.40） μg/L， P < 0.001）； （2） There was no statistically significant difference observed in the D-Lac treatment group compared to the control group ［Difference in LS Mean （95%CI） = 0.01 （0.00 ~ 0.02） mmol/L， P = 0.229］. However， subgroup analysis revealed that the decline in D-Lac levels was comparatively lower in the treatment group than in the control group （P = 0.043）， and there was an interaction between antibiotic use and the level of D-Lac decline within both groups （P for interaction = 0.012）； （3） The citrulline level was found to be significantly higher in the treatment group compared to the control group （P < 0.001）. There were no significant differences observed between the two groups regarding routine evaluation indexes， intestinal tolerance after enteral nutrition， and 90-day mortality rates （P > 0.05）. After a follow-up period of 90 days， it was noted that patients from the treatment group had a higher rate of early discharge when compared to those from the control group. （Breslow P = 0.012）. Conclusions Based on conventional evaluation criteria such as symptom remission， 90-day mortality， inflammatory markers， and amylase changes， probiotics do not demonstrate efficacy for severe pancreatitis. The dynamic changes of I-FABP and citrulline provide evidence supporting the potential of probiotics to reverse intestinal epithelial cell necrosis and promote recovery of their absorption/synthesis function in patients with severe pancreatitis. D-lactic acid has the potential to serve as an indicator for assessing the effectiveness of supplemental probiotics in the gut， thereby highlighting how the use of broad-spectrum antibiotics may limit their efficacy.