Pantethine, a natural low-molecular-weight thiol, shows broad activity in a large range of essential cellular pathways. It has been long known as a hypolipidemic and hypocholesterolemic agent. We showed recently that it exerts a neuroprotective action in mouse models of cerebral malaria and Parkinson's disease through multiple mechanisms. In the present study we looked at its effects on membrane lipid rafts that serve as platforms for molecules engaged in cell activity, therefore providing a target against inappropriate cell response leading to chronic inflammation. We found that pantethine-treated cells showed a significant change in raft fatty acid composition and cholesterol content, with ultimate downregulation of cell adhesion, CXCL12-driven chemotaxis and transendothelial migration of various T cell types, including human Jurkat cell line and circulating effector T cells. The mechanisms involved include the alteration of the following: i) CXCL12 binding to its target cells; ii) membrane dynamics of CXCR4 and CXCR7, the two CXCL12 receptors; iii) cell redox status, a crucial determinant in the regulation of the chemokine system. In addition, we considered the linker for activation of T cells (LAT) molecule to show that pantethine effects were associated with the displacement from the rafts of the acylated signaling molecules which palmitoylation level was reduced. In conclusion, the results presented here, together with previously published findings, indicate that, due to its pleiotropic action, pantethine can down-regulate the multifaceted process leading to pathogenic T cell activation and migration.