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Mutations in the GBA gene that encodes the lysosomal enzyme β-glucocerebrosidase (GCase) are a major genetic risk factor for Parkinson's disease (PD). In this study, we generated a set of differentiated and stable human dopaminergic cell lines that express the two most prevalent GBA mutations as well as GBA knockout cell lines as a in vitro disease modeling system to study the relationship between mutant GBA and the abnormal accumulation of α-synuclein. We performed a deep analysis of the consequences triggered by the presence of mutant GBA protein and the loss of GCase activity in different cellular compartments, focusing primarily on the lysosomal compartment, and analyzed in detail the lysosomal activity, composition, and integrity. The loss of GCase activity generates extensive lysosomal dysfunction, promoting the loss of activity of other lysosomal enzymes, affecting lysosomal membrane stability, promoting intralysosomal pH changes, and favoring the intralysosomal accumulation of sphingolipids and cholesterol. These local events, occurring only at a subcellular level, lead to an impairment of autophagy pathways, particularly chaperone-mediated autophagy, the main α-synuclein degradative pathway. The findings of this study highlighted the role of lysosomal function and lipid metabolism in PD and allowed us to describe a molecular mechanism to understand how mutations in GBA can contribute to an abnormal accumulation of different α-synuclein neurotoxic species in PD pathology., The authors wish to thank Dr. Arango (VHIR) for the PX461 vector and all the Vila lab members for their support. This work was supported by the Fondo de Investigación Sanitaria-Instituto de Salud Carlos III (Spain)-FEDER (PI17/00496 and PI20/00728), the Michael J. Fox Foundation, the Silverstein Foundation (MJFF 16182), and the BBVA Foundation (NanoERT). M.M. was supported by an FPU doctoral fellowship (FPU18/05595) from MINECO (Spain); J.R. was supported by a PERIS fellowship (Generalitat de Catalunya); E.P. was supported by a VHIR doctoral fellowship (VHIR, Barcelona).