Asparagine (Asn), an activator of ornithine decarboxylase (ODC), stimulates cell proliferation in intestinal epithelial cells. We hypothesized that Asn can mitigate LPS-induced injury of intestinal structure and barrier function by regulating inflammatory signaling pathways. We executed the following experiment using weanling pigs for each of the groups: (1) non-challenged control; (2) LPS-challenged control; (3) LPS + 0.5% Asn; (4) LPS + 1.0% Asn. After 21-d feeding, pigs received an i.p. injection of either saline or LPS. Four h after injection, the mid-jejunum and mid-ileum samples were collected. We found that Asn restored ODC expression that was decreased by LPS treatment. Asn also restored intestinal morphology and barrier function that were impaired by LPS treatment. In addition, Asn down-regulated intestinal caspase-3 protein expression and TNF-α concentration, and decreased the mRNA expression of intestinal TLR4, TLR4 downstream signals (myeloid differentiation factor 88, IL-1 receptor-associated kinase 1 and TNF-α receptor-associated factor 6 and NOD1, NOD2 and their adaptor molecule (receptor-interacting serine/threonine-protein kinase 2). Moreover, Asn decreased p38 phosphorylation but increased ERK1/2 phosphorylation. Our results suggest that Asn improves intestinal integrity during an inflammatory insult, which appears to be related to the decrease of intestinal pro-inflammatory cytokine (via TLR4, NODs and p38) and of enterocyte apoptosis (via p38 and ERK1/2).