Vascular smooth muscle cells (SMC) play a pivotal role in the pathogenesis of medial calcification (major cause of cardiovascular mortality in chronic kidney disease). We have recently demonstrated expression of the calcium-sensing receptor (CaSR) in human aortic SMC (HAoSMC) and human arteries and demonstrated a correlation between CaSR expression and medial calcification. The CaSR is involved in a number of diverse processes as hormone secretion, modulation of inflammation, proliferation, differentiation and apoptosis, however, functional significance of this receptor in vascular SMC is not fully understood. Here we examined CaSR-mediated intracellular signalling pathways and investigated the potential role of CaSR in regulating SMC proliferation and apoptosis. HAoSMC were incubated with CaSR agonists (neomycin and gentamycin) and signalling inhibitors. ERK1,2 activation was assessed by Western blot. Inositol triphosphate (IP3) production was measured using Biotrak assay (Amersham). Cell proliferation was determined by BrdU incorporation and apoptosis assessed by flow cytometry of propidium iodide stained cells. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Incubation of HAoSMC with 300µM neomycin resulted in 7.5-fold (p<0.05) increase in ERK1,2 phosphorylation. This induction was reduced (p<0.01) in the presence of 10µM PD98059 (MEK1 inhibitor), indicating that CaSR agonist-induced effects were mediated via classic MEK1/ERK1,2 pathway. ERK1,2 phosphorylation was almost completely abolished by 5µM U73122 (PLC inhibitor), indicating that PLC signalling was crucial for MEK1/ERK1,2 activation. No changes were observed with PI3K and PKC inhibitors. Confirming PLC activation, IP3 production was increased by neomycin/gentamycin (p<0.05) and reduced in the presence of U73122 (p<0.05). To confirm that ERK1,2 and PLC signalling were mediated via the CaSR, HAoSMC were transfected with CaSR siRNA. CaSR-knockdown resulted in attenuated ERK1,2 phosphorylation in response to neomycin (>50% of neomycin induction in control cells, p<0.01) while IP3 production was almost completely abolished. Treatment with neomycin increased HAoSMC proliferation >3-fold (p<0.01). This was reduced in CaSR-knockdown cells (p<0.01) and further inhibited by PD98059 and U73122 (p<0.05). Apoptosis was not affected by neomycin treatment or CaSR expression. However, inhibition of PLC signalling (incubation with U73122) produced a 3.5-fold increase in HAoSMC apoptosis (p<0.05), which was further increased by CaSR-knockdown (4.8-fold versus control siRNA, p<0.05). In conclusion, these data suggest that CaSR stimulation leads to activation of the MEK1/ERK1,2 and PLC pathways and increases cell proliferation. CaSR-mediated PLC activation is crucial for SMC survival and protection against apoptosis.