This study aimed to investigate the effects of porcine [gly2] glucagon-like peptide-2 (p[gly2]GLP-2) microspheres on lipopolysaccharide-challenged piglets and to evaluate efficacy of microspheres for administration compared with more conventional administration. Eighteen 21-d-old Duroc female piglets were randomly assigned into 3 groups: the control group (intraperitoneal injection with 3 mL saline solution daily), the glucagon-like peptide-2 (GLP-2) group (intraperitoneal injection with 3 mL p[gly2]GLP-2 at 20 nmol/kg BW daily), and the microsphere (MS) group (intraperitoneal injection with 100 mg p[gly2]GLP-2 microsphere suspension at Day 1). On Day 8, all piglets were injected with 100 μg lipopolysaccharide/kg BW. Results showed that administration of p[gly2]GLP-2 microspheres decreased the -lactic acid and methane dicarboxylic aldehyde content of the serum and increased the villus height and villus crypt ratio in the duodenum and ileum. Inducible nitric oxide synthase activity in the duodenum and ileum decreased, whereas enzyme activity for sucrose and Na-K adenosine triphosphatase in the ileum increased with treatment of p[gly2]GLP-2 microspheres. In the MS group, we observed downregulation of IL-8, TNF-α, IFN-γ, and GLP-2R mRNA expression in the ileum, upregulation of positive cell expression in the duodenum and positive cell expression in the ileum, and downregulation of GLP-2 receptor positive cell expression in the ileum. One injection of p[gly2]GLP-2 microspheres was as effective as p[gly2]GLP-2 administered for 7 d. Results suggested that p[gly2]GLP-2 can be a candidate agent for ameliorating weaning stress in piglets and that the use of microspheres is an ideal delivery system for GLP-2.