Originally considered an enigmatic polypeptide, the σ1 receptor has recently been identiﬁed as a unique ligand regulated protein. Many studies have shown the potential of σ1 receptor ligands for the treatment of various diseases of the central nervous system (CNS); nevertheless, almost no information about the 3D structure of the receptor and/or the possible modes of interaction of the σ1 protein with its ligands have been unveiled so far. With the present work we validated our σ1 3D homology model and assessed its reliability as a platform for σ1 ligand structure-based drug design. To this purpose, the 3D σ1 model was exploited in the design of 33 new σ1 ligands and in their ranking for receptor aﬃnity by extensive molecular dynamics simulation-based free energy calculations. Also, the main interactions involved in receptor/ligand binding were analyzed by applying a per residue free energy deconvolution and in silico alanine scanning mutagenesis calculations. Subsequently, all compounds were synthesized in our laboratory and tested for σ1 binding activity in vitro. The agreement between in silico and in vitro results conﬁrms the reliability of the proposed σ1 3D model in the a priori prediction of the aﬃnity of new σ1 ligands. Moreover, it also supports and corroborates the currently available biochemical data concerning the σ1 protein residues considered essential for σ1 ligand binding and activity.