Neuroblastoma (NB) is a pediatric malignancy originating from the neural crest cells of the sympathetic nervous system, and it often progresses aggressively due to the influence of epidermal growth factor (EGF). Our earlier research demonstrated that cathepsin D (CD) disrupts EGF-driven proliferation in NB cells cultured in 2D by inhibiting the EGFR/MAPK signaling pathway. While this highlights CD's regulatory function in tumor growth, its potential role in metastasis remains unexplored. In order to reproduce tumor heterogeneity, we engineered neuroblastoma (NB) clones with either silenced or overexpressed CD expression and co-cultured the clones in different proportions. We then analyzed the growth of the mixed populations in 2D (adherent) and 3D (suspension) culture conditions to mimic the stages of the metastatic process in response to EGF stimulation. We unexpectedly observed an opposite behavior in 2D and 3D cells with different levels of CD. In particular, cells overexpressing CD demonstrated a greater adaptability to growth in suspension cultures. In contrast, cells with suppressed CD expression exhibited enhanced growth under adherent conditions in two-dimensional environments. Different CD levels lead to different behaviors even during the transition from 3D to 2D: cells that overexpress CD demonstrated an increase in N-cadherin levels, while cells with silenced CD showed a greater propensity to revert to a mesenchymal-to-epithelial-like phenotype, as evidenced by the elevated expression of E-cadherin. The contrasting roles of CD in driving cancer cell growth under 2D and 3D conditions indicate that clonal evolution may favor the emergence of subpopulations with distinct CD levels, optimizing their adaptability to various metastatic niches. This highlights the potential epigenetic regulation of CD as a mechanism supporting survival and proliferation in both substrate-adherent and neurosphere-forming NB cells. Consequently, disrupting the epigenetic control of CD could represent a promising therapeutic approach to hinder NB progression and metastasis.