The immune function of the chicken spleen depends on its different compartments of red and white pulps, but little is known about the mechanism underlying lymphocyte homing towards the different compartments. In the present study, the role of lymphocyte homing in the chicken spleen was investigated during lipopolysaccharide (LPS) stimulation. Morphological analysis demonstrated the cuboidal endothelial cells of the splenic sheathed capillary facilitated the passage of lymphocyte homing to the chicken spleen. The tissue-specific adhesion molecules-vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MADCAM-1) expressed on the sheathed capillary, which suggested the high endothelial venule (HEV)-like vessels of the chicken spleen. Electron microscope analysis showed LPS activated the endothelium of the sheathed capillary and recruited lymphocytes to the chicken spleen. Transferring of 5, 6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeled lymphocytes depicted the rout of lymphocyte homing to the compartments of the chicken spleen was from the white pulp to the red pulp. Furthermore, the mRNA and protein levels of adhesion molecular integrin beta 1 and VCAM-1 increased after LPS stimulation. The mechanism underlying the integrin beta 1 and VCAM-1 during LPS stimulation might be associated with the integrin linked kinase (ILK)-dependent regulation of protein kinase B (PKB/AKT). This study firstly shows lymphocyte homing in the chicken spleen after LPS-induced inflammation. These results contribute to our knowledge of comparative immunology and provide a better means for investigating the pharmacological strategies concerning the possible role of lymphocyte homing in inflammation and immunological reactions in infectious disease.