Long-lived PFKFB3-expressing β-cells are dysfunctional partly because of prevailing glycolysis that compromises metabolic coupling of insulin secretion. Their accumulation in type 2 diabetes (T2D) appears to be related to the loss of apoptotic competency of cell fitness competition that maintains islet function by favoring constant selection of healthy winner cells. To investigate how PFKFB3 can disguise the competitive traits of dysfunctional loser β-cells, we analyzed the overlap between human β-cells with bona fide loser signature across diabetes pathologies using the HPAP scRNA-seq and spatial transcriptomics of PFKFB3-positive β-cells from nPOD T2D pancreata. The overlapping transcriptional profile of loser β-cells was represented by down-regulated ribosomal biosynthesis and genes encoding for mitochondrial respiration. PFKFB3-positive loser β-cells had the reduced expression of HLA class I and II genes. Gene-gene interaction analysis revealed that PFKFB3 rs1983890 can interact with the anti-apoptotic gene MAIP1 implicating positive epistasis as a mechanism for prolonged survival of loser β-cells in T2D. Inhibition of PFKFB3 resulted in the clearance of dysfunctional loser β-cells leading to restored glucose tolerance in the mouse model of T2D.