Wolfram syndrome is caused by mutations in WFS1 encoding wolframin, a polytopic membrane protein of the endoplasmic reticulum. Here, we investigated the molecular pathomechanisms of four missense and two truncating mutations in WFS1. Expression in COS-7 cells as well as direct analysis of patient cells revealed that WFS1 mutations lead to drastically reduced steady-state levels of wolframin. All mutations resulted in highly unstable proteins which were delivered to proteasomal degradation. No wolframin aggregates were found in patient cells suggesting that Wolfram syndrome is not a disease of protein aggregation. Rather, WFS1 mutations cause loss-of-function by cellular depletion of wolframin.