The opioid peptide β-endorphin coexists in the pituitary and brain in its -acetylated form, which does not bind to opioid receptors. We now report that these neuropeptides exhibited opposite effects in in vivo paradigms, in which ligands of the sigma type 1 receptor (σ1R) displayed positive effects. Thus, -acetyl β-Endorphin reduced vascular infarct caused by permanent unilateral middle cerebral artery occlusion and diminished the incidence of -methyl-D-aspartate acid-promoted convulsive syndrome and mechanical allodynia caused by unilateral chronic constriction of the sciatic nerve. Moreover, -acetyl β-Endorphin reduced the analgesia of morphine, β-Endorphin and clonidine but enhanced that of DAMGO. All these effects were counteracted by β-Endorphin and absent in σ1R mice. We observed that σ1Rs negatively regulate mu-opioid receptor (MOR)-mediated morphine analgesia by binding and sequestering G proteins. In this scenario, β-Endorphin promoted the exchange of σ2Rs by G proteins at σ1R oligomers and increased the regulation of G proteins by MORs. The opposite was observed for the -acetyl derivative, as σ1R oligomerization decreased and σ2R binding was favored, which displaced G proteins; thus, MOR-regulated transduction was reduced. Our findings suggest that the pharmacological β-Endorphin-specific epsilon receptor is a σ1R-regulated MOR and that β-Endorphin and -acetyl β-Endorphin are endogenous ligands of σ1R.