The amyloid-cascade hypothesis proposes that the main protagonist in Alzheimer’s disease is oligomeric amyloid-β (Aβ). Protofibrillar Aβ1-42 induces cytotoxicity through various signalling cascades, including calcium dysregulation via modulation of neuronal voltage-gated calcium channels (VGCCs). The aim of this work was to examine the role of protofibrillar Aβ1-42 in modulating VGCCs present in SH-SY5Y cells. Within undifferentiated cells, Cav1.2, Cav1.3, Cav2.2 and Cav3.1 were detected at gene level (qPCR). Upon retinoic acid (10μM; 3 days) differentiation of SH-SY5Y cells, a reduction in Cav3.1 was observed. No gene expression of Cav1.1, Cav1.4, Cav2.1, Cav2.3, Cav3.2 and Cav3.3 was detected in SH-SY5Y of either differentiation states. Prior to experimental use, Aβ1-42 aggregation status was verified via transmission electron microscopy. A time-dependent (0-48h) formation of Aβ1-42 protofibrils (>100nm in length) was observed using NH4OH pre-treatment. Protofibrillar Aβ1-42 (24h) induced neurotoxicity (XTT assays) in undifferentiated SH-SY5Y cells, in a concentration-dependent manner. A significant reduction in viability was observed with 3μM (21.2±6.1%; n=11; P<0.05) and 10μM Aβ1-42 (23.9±6.8%; n=8; P<0.01) treatment. In contrast, lower concentrations of 1nM (1.8±6.8%; n=8; P=1.0), 100nM (3.7±5.5%; n=16; P=0.9) and 1μM Aβ1-42 (2.7±5.4%; n=17; P=1.0) did not induce cytotoxicity. In parallel, PCR gene expression analysis revealed that treatment with 100nM Aβ1-42 induced a significant reduction in Cav1.3 expression (0.81±0.04; n=5; 24h; P<0.01) within differentiated cells. Additionally, following 1μM Aβ1-42 treatment, an increase in Cav1.2 expression (1.8±0.15; n=3; 24h; P<0.05) was also seen. No significant change in expression was observed within undifferentiated SH-SY5Y cells treated with either 100nM or 1μM Aβ1-42 treatment (24h). These data demonstrate differential Aβ1-42 concentration-dependent modulation of VGCCs selectively within differentiated SH-SY5Y cells. Whole-cell patch clamp studies using Cav2.2 (α1B(CaV2.2)/β1b/α2δ) stably-transfected HEK293 cells revealed changes in the voltage-dependence of activation (slope factor k) (-7.4±3.1mV; n=15; P<0.05) with 100nM Aβ1-42 treatment. Current amplitude and other biophysical properties (Gmax, Rp and Vhalf), as calculated by Boltzmann function fitting, did not significantly alter. Collectively, our work demonstrates that protofibrillar Aβ1-42 can induce neurotoxicity, and modulate VGCCs through changes at both the molecular and functional level. Our work will aid the understanding of amyloid pathology, and highlight the potential to target VGCCs to suppress Aβ disease mediated pathology.