The genomic activity of 1,25(OH)D is mediated by vitamin D receptor (VDR), whilst non-genomic is associated with protein disulfide isomerase family A member 3 (PDIA3). Interestingly, our recent studies documented that PDIA3 is also involved, directly or indirectly, in the modulation of genomic response to 1,25(OH)D. Moreover, PDIA3 was also shown to regulate cellular bioenergetics, possibly through the modulation of STAT signaling. Here, the role of VDR and PDIA3 proteins in membrane response to 1,25(OH)D and calcium signaling was investigated in squamous cell carcinoma A431 cell line with or without the deletion of and genes. Calcium influx was assayed by Fura-2AM or Fluo-4AM, while calcium-regulated element (NFAT) activation was measured using a dual luciferase assay. Further, the levels of proteins involved in membrane response to 1,25(OH)D in A431 cell lines were analyzed via Western blot analysis. The deletion of either or resulted in the decreased baseline levels of Ca and its responsiveness to 1,25(OH)D; however, the effect was more pronounced in A431∆. Furthermore, the knockout of either of these genes disrupted 1,25(OH)D-elicited membrane signaling. The data presented here indicated that the VDR is essential for the activation of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), while PDIA3 is required for 1,25(OH)D-induced calcium mobilization in A431 cells. Taken together, those results suggest that both VDR and PDIA3 are essential for non-genomic response to this powerful secosteroid.