Rationale: Hyperhomocysteinemia contributes to vascular dysfunction and risks of cardiovascular diseases. Stromal cell–derived factor (SDF)-1, a chemokine expressed by endothelial cells (ECs), is highly expressed in advanced atherosclerotic lesions. The interplays among homocysteine, chemokines, and shear stress in regulating vascular endothelial function are not clearly understood. Objective: To investigate the mechanisms for modulations of EC SDF-1 expression by homocysteine and shear stress. Methods and Results: Homocysteine stimulation induced dose- and time-dependent SDF-1 expression and phosphorylation of mitogen-activated protein kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. By using specific inhibitors, small interfering (si)RNA, and dominant negative mutants, we demonstrated that activation of JNK pathway is critical for the homocysteine-induced SDF-1 expression. Transcription factor ELISA and chromatin immunoprecipitation assays showed that homocysteine increased Sp1- and AP-1–DNA binding activities in ECs. Inhibition of Sp1 and AP-1 activations by specific siRNA blocked the homocysteine-induced SDF-1 promoter activity and expression. Preshearing of ECs for 1 to 4 hours at 20 dyn/cm2 inhibited the homocysteine-induced JNK phosphorylation, Sp1 and AP-1 activation, and SDF-1 expression. The homocysteine-induced SDF-1 expression was suppressed by NO donor. Inhibitor or siRNA for endothelial NO synthase abolished the shear inhibition of SDF-1 expression. Conclusions: Our findings serve to elucidate the molecular mechanisms underlying the homocysteine induction of SDF-1 expression in ECs and the shear stress protection against this induction.