Purpose <p>The cellular mechanisms underlying tolerance development to psychostimulant-induced neurotoxicity remain poorly understood. This study investigated these mechanisms using pyrrolidinophenone derivatives (PPs), potent amphetamine-type stimulants with strong dopaminergic activity and high cytotoxicity, aiming to establish a neuronal tolerance model and to explore adaptive processes relevant to substance use disorder.</p> Methods <p>Human SK-N-SH neuronal cells were chronically exposed to α-pyrrolidinooctanophenone (α-POP) to prepare the drug-resistant cell line, SH/POP. Transcriptomic profiling was performed to identify gene expression alterations associated with tolerance development.</p> Results <p>Cell sensitivity assay showed that SH/POP cells can survive at lethal concentrations (&gt; 40 μM) of α-POP. RNA sequence analysis of the resistant cells identified alterations in 1,298 differentially expressed genes and the gene ontology analysis surmised an upregulation of calcium-binding-related genes. The development of α-POP resistance reduced the basal Ca²⁺ concentration and suppressed the caspase-3 activation elicited by the drug. Additionally, the development down-regulated the phosphorylation of a transcription factor cAMP response element-binding protein (CREB) and expressions of CREB-target genes, Fos proto-oncogene AP-1 transcription factor subunit and neurotensin. Furthermore, constitutive activation of endoplasmic reticulum stress responses and selective enhancement of trypsin-like proteasome activity in SH/POP cells were detected.</p> Conclusions <p>Resistance development of neuronal cells to PPs is ascribable to suppression of calcium-dependent apoptosis and CREB signaling, and constitutive activation of endoplasmic reticulum stress responses. The SH/POP cell line represents a novel in vitro model to study molecular adaptations to psychostimulant toxicity and provides insights into neuroadaptive mechanisms underlying substance use disorder.</p>