Glucosylceramide (GC) is a metabolic intermediate derived from the cellular turnover of membrane gangliosides and globosides. The catabolism of GC is impaired in Gaucher disease (GD) and consequently GC accumulates in affected cells leading to clinical manifestations of GD. The primary cell type affected in GD is the macrophage, and we investigated what effect excess GC has on the spatial coordination of other sphingolipids and phospholipids in a macrophage model of GD. A THP-1 macrophage model of GD was established by supplementation of the culture media with conduritol B epoxide, a specific irreversible inhibitor of acid beta-glucosidase. This cell model accumulated up to 12-fold more GC compared with untreated cells. Sub-cellular fractionation showed that, initially, the primary site of GC accumulation was the lysosome but as more GC accumulated it distributed relatively evenly across the cell and was present in all sub-cellular fractions. We also observed secondary elevations in the concentrations of ceramide, di- and trihexosylceramide and phosphatidylglycerol, which all had similar sub-cellular distributions to that of GC, initially increasing in the lysosome and then throughout the sub-cellular compartments. Our results suggest that with excess GC accumulation, the pathway trafficking GC to the lysosome becomes saturated and GC as well as other sphingolipids are shunted to other parts of the cell. The presence of these sphingolipids at non-physiological concentrations is likely to interfere with other biochemical pathways outside the lysosome, leading to cell dysfunction and ultimately pathological mechanisms apparent in GD, such as macrophage activation.