Leukocyte recruitment to the inflammation site is a multi‐step process governed by specific signalling cascades. After adhesion in the lumen, leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac‐1‐dependent manner. The signalling mechanisms that regulate post‐adhesion steps of intraluminal crawling, transmigration and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various steps of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient in microvasculature of murine cremaster muscle using intravital microscopy and time‐lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, transmigration time and detachment time, and impaired the initiation of crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac‐1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.