The efficient conversion of lignocellulosic biomass is at the core of ensuring the economic feasibility of biorefineries, where the comprehensive utilization of xylose serves as a determinant of overall conversion efficiency. Pichia kudriavzevii has demonstrated significant potential in industrial bioprocessing owing to its tolerance to low pH, high temperatures, and environmental stressors. However, its innate deficiency in xylose assimilation severely restricts its application in biomass valorization. [Objective] To elucidate the molecular mechanisms underlying the silencing of xylose metabolism in P. kudriavzevii E1, thereby providing a theoretical basis for the bioconversion of lignocellulosic feedstocks. [Methods] The xylose assimilation capacity of P. kudriavzevii E1 was evaluated, and its genome was analyzed to identify the genes and metabolic bottlenecks associated with xylose assimilation. Comparative transcriptomics was employed to characterize the differential expression of metabolic genes before and after the introduction of a xylose transporter. Furthermore, the heterologous expression of genes involved in efficient xylose metabolism was performed to verify specific rate-limiting steps within the pathway. [Results] Bioinformatics analysis, coupled with the functional restoration of xylose uptake via heterologous transporter expression, confirmed that the lack of high-affinity xylose transporters was the primary limiting factor for xylose assimilation in P. kudriavzevii E1. Although three genes—PkXYL1, PkXYL2, and PkXKS1—encoding core enzymes of the xylose redox pathway were natively present in the P. kudriavzevii E1 genome, in vitro enzymatic assays revealed that the low relative activity of PkXR was a critical cause of substrate accumulation and slow xylose metabolism. Comparative transcriptomics of the engineered strain P. kudriavzevii E1-Xpg4562 indicated that yeast cells underwent profound metabolic reprogramming in xylose-containing media, preferentially activating ribosome biogenesis and oxidative phosphorylation. However, the significant downregulation of TAL1 and the insufficient transcriptional response of genes in the pentose phosphate pathway (PPP) resulted in inefficient PPP flux. This prevented the effective redirection of carbon flux into glycolysis, thereby obstructing downstream xylose metabolism. Finally, quantification of the expression of key xylose metabolism genes identified by transcriptomics further demonstrated that the uncoordinated transcriptional regulation of essential downstream genes hindered overall metabolic efficiency. [Conclusion] The silencing of xylose metabolism in P. kudriavzevii E1 results from the combined effects of deficient substrate transport, low endogenous catalytic activity, and uncoordinated transcriptional regulation. This study provides a crucial theoretical foundation for the precision engineering of xylose metabolic pathways in non-conventional industrial yeasts.