Yarrowia lipolytica holds significant promise for bioconversion of renewable feedstocks, yet its capacity to efficiently assimilate mannitol, a major carbon source in olive mill wastewater (OMWW), remains limited. Here, we conducted adaptive laboratory evolution (ALE) under mannitol-exclusive conditions using four different Y. lipolytica strains followed by integrative genetic and transcriptomic analyses. These strains exhibited markedly enhanced mannitol assimilation and growth of upwards of 22-fold increase in utilization. Whole-genome sequencing and reverse engineering revealed that a point mutation in STA1 (A403T), encoding glucan 1,4-α-glucosidase, contributed to improved growth. Transcriptomic analysis showed upregulation of MFE2, associated with peroxisomal β-oxidation, and downregulation of DGA1, a key gene for triacylglycerol synthesis, indicating a systems-level metabolic shift that favors energy mobilization over storage lipid accumulation. When cultivated in OMWW-derived media, the evolved strains achieved two-fold higher lipid production compared to its parental strain from mannitol and simultaneously reduced the chemical oxygen demand (COD) by 90 %. Taken together, these findings expand the genetic and regulatory landscape underlying mannitol assimilation in Y. lipolytica and demonstrate the potential of evolved strains for bioconversion of waste-derived substrates into valuable biochemicals.