Summary. Background: Our previous results demonstrated that aggregated low density lipoprotein (agLDL) induces tissue factor (TF) expression and activation through Rho A translocation in human vascular smooth muscle cells (VSMC). We also previously demonstrated that membrane sphingomyelin (SM) content is higher in agLDL-exposed VSMC than in control cells. The main enzymes regulating cellular SM content are the family of sphingomyelinases (Smases) that hydrolize SM to phosphorylcholine and ceramide (CER). Objectives: We wished to investigate whether agLDL has the ability to modulate acidic- (A-) and neutral (N-) Smase activity and whether or not this effect is related to the upregulatory effect of agLDL on Rho A translocation and TF activation in human VSMC. Methods and Results: By measuring generated [14C]-phosphorylcholine, we found that agLDL significantly decreased A-Smase and specially N-Smase activity. Pharmacological Smase inhibitors increased Rho A and TF. Specific loss-of-function of A-Smase or N-Smase 1 (N1-Smase) by siRNA treatment (500 nmol L−1, 12 hours) dramatically increased membrane Rho A protein levels (5- and 3-fold, respectively). Concomitantly, TF protein expression and TF procoagulant activity were also increased. Inhibition of A-Smase or N-Smase activity by agLDL, siRNA-anti A- or N1-Smase or pharmacological treatment significantly increased the SM content of vascular cells. The inhibition of SM synthesis by fumonisin B1 (FB1) prevented the upregulatory effect of agLDL on TF. Conclusions: These results demonstrate that inhibition of both A- and N1-Smase might explain the upregulatory effect of agLDL on TF activation, and suggest that this effect is related, at least in part, to membrane SM enrichment.