Voltage-dependent Ca²⁺ channels (VDCC) play a vital role in cell control mechanisms. In this study we profiled the expression of VDCC genes, and their function, in human embryonic stem cells (hESC) during PAX4-directed differentiation toward the pancreatic β-cell lineage. H7 hESC clones stably-transfected with PAX4 (H7.Px4) and untransfected H7 controls (H7) were allowed to spontaneously differentiate over a 3-week period following embryoid body (EB) formation. Gene expression was assessed using standard RT-PCR and SYBR-green quantitative PCR (Q-PCR) at days 0, 7, 14 and 21 following initiation of differentiation. Physiological function was assessed using Ca²⁺ microfluorimetry of Fura2-loaded EBs challenged with depolarising concentrations of KCl (40 mM) at days 7, 14 and 21 of differentiation. RT-PCR revealed the expression of transcripts for CACNA1S, CACNA1C, CACNA1D, CACNA1A, CACNA1B and CACNA1E, and CACNB1 to 4 in EBs from H7 and H7.Px4 and also in human foetal pancreas, although neither CACNA1S or CACNA1E were expressed in human neonatal pancreas. Interestingly, Q-PCR demonstrated significant upregulation of CACNA1A and CACNA1C expression at all time points in the H7.Px4 EBs whereas CACNA1D expression was only transiently upregulated in these EBs relative to H7 control EBs. The increase in VDCC gene expression over time correlated with an enhanced responsiveness of EBs to 40 mM KCl-induced Ca²⁺ signalling in the H7.Px4 EBs relative to H7 control EBs. In control EBs, intracellular Ca²⁺ responses ([Ca²⁺]_i) to KCl stimulation were poor between days 7 and 21 of differentiation (e.g. 9/31 EBs responded at day 21) although the size of responses increased over time (16±2 nM; n=11 at day 7 vs 39±15 nM; n=9 at day 21). In comparison, the numbers of H7.Px4 EBs responding to KCl stimulation were significantly higher (e.g. 34/34 EBs responded at day 21) with large changes in [Ca²⁺]_i observed from 7 days onwards (e.g. 36±4 nM at day 7, n=13/27). In summary, PAX4 overexpression in hESC EBs led to significant changes in intracellular Ca²⁺ signaling. These changes could be explained by upregulated expression of VDCC subunit transcripts. Since constitutive PAX4 expression also leads to expression of transcriptional and functional markers of insulin-secreting cells, these data provide additional support for the use of PAX4 as a lineage determinant of β-cells in hESC.