The use of total parenteral nutrition (TPN) in the treatment of critically ill patients has been the subject of debate because it has been associated with disturbances in intestinal homeostasis. Important factors in maintaining the intestinal homeostasis are the intestinal microbiota and Paneth cells, which exist in a mutually amendable relationship. We hypothesized that the disturbed intestinal homeostasis in TPN-fed individuals results from an interplay between a shift in microbiota composition and alterations in Paneth cells. We studied the microbiota composition and expression of Paneth cell antimicrobial proteins in rats receiving TPN or a control diet for 3, 7, or 14 d. qPCR analysis of DNA extracts from small intestinal luminal contents of TPN-fed rats showed a shift in Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes after 14 d (P < 0.05) compared with the control group. This finding coincided with greater staining intensity for lysozyme and significantly greater mRNA expression of Paneth cell antimicrobial proteins lysozyme (P < 0.05), rat alpha-defensin 5 (P < 0.01), and rat alpha-defensin 8 (P < 0.01). Finally, 14 d of TPN resulted in greater circulating ileal lipid-binding protein concentrations (P < 0.05) and greater leakage of horseradish peroxidase (P < 0.01), which is indicative of enterocyte damage and a breached intestinal barrier. Our findings show a shift in intestinal microbiota in TPN-fed rats that correlated with changes in Paneth cell lysozyme expression (r(s) = -0.75, P < 0.01). Further studies that include interventions with microbiota or nutrients that modulate them may yield information on the involvement of microbiota and Paneth cells in TPN-associated intestinal compromise.