The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca2+ dynamics were examined in WFS1‐knockdown and ‐overexpressing HEK293 cells. Studies using ER‐targeted Ca2+‐sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca2+ levels by increasing the rate of Ca2+ uptake. Furthermore, Ca2+ imaging with Fura‐2 showed the magnitude of the store‐operated Ca2+ entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca2+ homeostasis, at least partly, by modulating the filling state of the ER Ca2+ store.