<p>Lysosomal acid lipase (LAL) plays essential roles in bone metabolism and stem/progenitor cell behaviours. Given its established role in skeletal and periodontal biology, the present study aims to determine the specific role of LAL in regulating the critical behaviours of dental pulp stem cells (DPSCs), including cell viability, proliferation, apoptosis, and differentiation ability. Cells were treated with a common LAL inhibitor, Lalistat-2. Cell viability was determined using a live/dead assay. Cell proliferation was examined using the MTT and colony-forming assays. Cell cycle progression and apoptosis were investigated. The in vitro mineralisation and intracellular lipid accumulation were detected using Alizarin Red S and Oil Red O staining, respectively. mRNA expression was examined by real-time polymerase chain reaction. A high-throughput RNA sequencing technique was used to profile mRNA expression. Lalistat-2 treatment dysregulated mRNA expression in DPSCs, with pathway analysis identifying changes in several KEGG pathways, including DNA replication, the Hippo pathway, the cell cycle, and the TGF-β signalling pathway. Lalistat-2 was non-toxic at low concentrations (1–50 µM), though 50 µM significantly impaired proliferation and colony formation by inducing apoptosis without altering overall cell cycle progression. Lalistat-2 had complex effects on differentiation: low concentrations (1–10 µM) enhanced in vitro mineralisation, while it consistently reduced adipogenic differentiation by decreasing lipid accumulation and marker gene expression. In conclusion, Lalistat-2 treatment induces apoptosis and suppresses adipogenic differentiation in DPSCs. Notably, low concentrations enhanced in vitro mineralisation, highlighting a complex, concentration-dependent effect on differentiation. These findings confirm LAL’s critical role in DPSCs’ homeostasis and regeneration and underscore the importance of their metabolic adaptability.</p>