Human d-amino acid oxidase (EC 22.214.171.124; hDAAO) is a peroxisomal flavoenzyme significantly enriched in the mammalian brain. hDAAO has been proposed to play (with serine racemase; EC 126.96.36.199) an essential role in the catabolism of d-serine, an ‘atypical’ key signalling molecule that acts as allosteric activator of the N-methyl-d-aspartate-type glutamate receptor (NMDAr). hDAAO and its interacting partner pLG72 have been related to schizophrenia, a highly disabling psychiatric disorder in which a dysfunction of NMDA-mediated neurotransmission is widely assumed to occur. We previously demonstrated that the d-serine cellular concentration depends on hDAAO and pLG72 expression levels and that newly-synthesized hDAAO interacts with its modulator in the cytosol, being progressively destabilized and inactivated. To obtain insight into the mechanisms regulating cellular d-serine levels, we investigated the degradation pathways of hDAAO and pLG72 in U87 glioblastoma cells stably expressing enhanced yellow fluorescent protein-hDAAO (peroxisomal), hDAAO-enhanced yellow fluorescent protein (cytosolic) or pLG72-enhanced cyan fluorescent protein (mitochondrial) proteins. hDAAO is a long-lived protein: the peroxisomal fraction of this flavoprotein is degraded via the lysosomal/endosomal pathway (and blocking this pathway increases the cellular hDAAO activity and decreases d-serine levels), whereas the cytosolic portion is ubiquitinated and targeted to the proteasome. By contrast, pLG72 shows a rapid turnover (t1/2 ≈ 25–40 min) and is degraded via the proteasome system, albeit not ubiquitinated. Overexpression of pLG72 increases the turnover of hDAAO, in turn playing a protective role against excessive d-serine depletion.