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.