The sigma-1 receptors bind diverse kinds of psychoactive compounds, including cocaine, and translocate upon stimulation by these compounds. However, the exact intracellular localization and dynamics of sigma-1 receptors have been unclear. We recently found that sigma-1 receptors specifically localize on cholesterol-enriched loci on the endoplasmic reticulum (ER) membrane that function as neutral lipid storage sites (i.e., ER lipid droplets or ER-LDs) from which neutral lipids bud out to form cytosolic lipid droplets. By combining immunocytochemistry and real-time monitoring of enhanced yellow fluorescent protein (EYFP)-tagged sigma-1 receptors (Sig-1R-EYFP) in living cells, we characterized the sigma-1 receptor translocation in this study. (+)-Pentazocine, a selective sigma-1 receptor agonist, causes a significant decrease of sigma-1 receptors in ER-LDs and a diffused distribution of sigma-1 receptors over the entire endoplasmic reticulum reticular network in NG108-15 cells. In the presence of sigma-1 receptor agonists, Sig-1R-EYFP move out from ER-LDs and slide along the endoplasmic reticulum network toward nuclear envelope and the tip of neurites. Fluorescence recovery after photobleaching analysis demonstrates that Sig-1R-EYFP on endoplasmic reticulum reticular network are highly mobile compared with those in ER-LDs. A sucrose gradient fractionation study shows that (+)-pentazocine shifts sigma-1 receptors from ER-LD membranes to higher density membranes. These results indicate that sigma-1 receptors localize on ER-LDs and upon stimulation translocate on continuous endoplasmic reticulum reticular network toward peripheries of cells. Because sigma-1 receptors specifically target ER lipid storage sites and compartmentalize neutral lipids therein, these results suggest that sigma-1 receptors' dynamic translocation might affect lipid transport and distribution in neuronal cells.