Abstract: Type 2 diabetes (T2D) is associated with progressive dysfunction of β-cell but how disease-associated transcriptional changes converge into biochemical patterns remains incompletely defined. This study aimed to identify and prioritize late-T2D-associated biochemical alterations and candidate mechanisms in β-cells by integrating donor-level transcriptomics, biochemical and mechanistic prioritization, pathway analysis, independent human β-cell validation, and genetic anchoring. β-cell profiles from the human pancreatic islet dataset GSE221156 were aggregated at the donor level and analyzed across the three prespecified pairwise comparisons of non-diabetic (ND), pre-diabetic (PD), and T2D states. Transcriptional patterns were classified for their disease-stage association along the ND–PD–T2D sequence, and then biochemical and mechanistic prioritization and functional enrichment were performed. An 18-gene Core-18 subset was defined before independent validation. Genetic anchoring was performed using T2D Knowledge Portal effector genes. No significant differential expression was found between ND and PD, but 266 genes showed differential expression between PD and T2D, and 518 genes showed differential expression between ND and T2D. 154 genes were classified as Late-T2D-associated and 364 as Overall-T2D-associated. 55 candidates genes were prioritized across seven mechanistic axes, and an 18-gene Core-18 subset was identified. Functional enrichment identified 12 FDR-significant enrichment terms, involving mostly GPCR- and peptide-receptor-related signaling, β-cell development and gene regulation, corresponding to 23 unique genes in 83 pathway–gene records. Fourteen of 18 Core-18 candidates were directionally concordant, with 12 directionally supported candidates and 2 lower-confidence candidates. Genetic anchoring revealed HNF1A and SLC2A2 as shared genes between the pathway-associated gene set and the T2D Knowledge Portal effector-gene set, with significant genetic over-representation in two β-cell regulatory pathways. The study highlights a late-T2D-associated β-cell biochemical profile that includes extracellular signaling, metabolic sensing, β-cell regulatory mechanisms, and biochemical control layers. Directional and genetic evidence was integrated to prioritize candidate pathways and genes for mechanistic follow-up.