Integrin-mediated rolling and firm cell adhesion are two critical steps in leukocyte trafficking. Integrin alpha 4 beta 1 mediates a mixture of rolling and firm cell adhesion on vascular cell adhesion molecule-1 (VCAM-1) when in its resting state but only supports firm cell adhesion upon activation. The transition from rolling to firm cell adhesion is controlled by integrin activation. Kindlin-3 has been shown to bind to integrin tails and trigger integrin activation via inside-out signaling. However, the role of kindlin-3 in regulating resting alpha 4 beta 1-mediated cell adhesion is not well characterized. Herein we demonstrate that kindlin-3 was required for the resting alpha 4 beta 1-mediated firm cell adhesion but not rolling adhesion. Knockdown of kindlin-3 significantly decreased the binding of kindlin-3 to beta 1 and down regulated the binding affinity of the resting alpha 4 beta 1 to soluble VCAM-1. Notably, it converted the resting alpha 4 beta 1-mediated firm cell adhesion to rolling adhesion on VCAM-1 substrates, increased cell rolling velocity, and impaired the stability of cell adhesion. By contrast, firm cell adhesion mediated by Mn2+ activated alpha 4 beta 1 was barely affected by knockdown of kindlin-3. Structurally, lack of kindlin-3 led to a more bent conformation of the resting alpha 4 beta 1. Thus, kindlin-3 plays an important role in maintaining a proper conformation of the resting alpha 4 beta 1 to mediate both rolling and firm cell adhesion. Defective kindlin-3 binding to the resting alpha 4 beta 1 leads to a transition from firm to rolling cell adhesion on VCAM-1, implying its potential role in regulating the transition between integrin-mediated rolling and firm cell adhesion.