Objective To elucidate the spreading dynamics of β2 integrin expressed human neutrophils (PMNs) on ICAM-1-immobilized substrate. Methods The fraction of PMN spreading on the substrate pre coated by 10, 20, or 100 μg/mL intercellular adhesive molecule-1 (ICAM-1) was quantified when that on 2% human serum albumin (HSA) immobilized or that on blank substrate was served as control. The site density of β2 integrin expressing on PMNs was determined using flow cytometry and the regulation of β2 integrin subunits was defined using the fraction of PMN spreading on 100 μg/mL ICAM-1 substrate by blocking CD11a or CD11b subunit of β2 integrin. Results PMN spreading was presented on ICAM-1-immobilized substrate but absent on 2% HSA-immobilized substrate, supporting the specificity of β2 integrin induced spreading. Time course of neutrophil spreading on ICAM-1 substrate was density dependent of both ICAM-1 and β2 integrin molecules. The fraction of PMN spreading was reduced significantly when the expression of CD11b subunit was blocked. Conclusions PMN spreading was mediated specifically by β2 integrin-ICAM-1 interactions and determined by the expression of β2 integrin and ICAM-1, in which CD11b subunit played a dominate role.