Shear stress (SS), the tangential component of hemodynamic forces, modulates the expression of several genes in endothelial cells. However, no information is available about its effect on chromatin structure, which plays a key role in gene transcription. In this study, a link between SS and chromatin remodeling was established in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to SS of 10 dyne/cm² per second, in the presence or absence of the histone deacetylase inhibitor trichostatin A, and assayed for histone H3 and histone H4 modifications. SS induced histone H3 serine phosphorylation at position 10 (S10) and lysine acetylation at position 14 (K14) but required trichostatin A to induce H3 phosphoacetylation and H4 acetylation. The phosphatidylinositol 3-kinase inhibitor wortmannin and the mitogen-activated protein kinase inhibitor PD98059 decreased SS-dependent histone H3 phosphorylation, without affecting its acetylation; the p38 inhibitor SB203580 reduced both H3 phosphorylation and acetylation, whereas the protein kinase A inhibitor PKI-tide reduced histone H3 acetylation. Remarkably, the abrogation of histone acetylation inhibited SS-dependent c-fos expression. SS also activated ribosomal S6 kinase-2 and mitogen- and stress-activated kinase-1 protein kinases and promoted the formation of a cAMP-responsive element–binding protein (CREB)/CREB-binding protein complex, providing the molecular basis for the increase in histone acetyltransferase activity observed in HUVECs exposed to SS. Finally, the effect of SS on chromatin remodeling was examined. In HUVECs exposed to SS, chromatin within c-fos and c-jun promoters was specifically immunoprecipitated by an antibody against acetylated histone H3 on K14. These results indicate that SS induces posttransduction modifications of histones; this is an early step toward the flow-dependent regulation of gene expression.