Diabetes Mellitus is a complex metabolic disorder with one of the highest prevalence rates in the world. The present study probes into the Thr-Ala 642 mutation of Akt substrate of 160 kDa (AS160) which has been implicated in diabetes by the dysregulation of glucose transported vesicle 4 (GLUT4) translocation. Our study provides a possible evidence on structural basis dysfunction of AS160 and how the association of phosphorylated AS160 with 14-3-3, a downstream binding partner regulating GLUT4 translocation got disrupted due to T642A mutation. We initially derived the disease-causing mutation (Thr642Ala) among others through in-silico based statistical analysis. Subsequently, we interpreted the perturbation induced in the structural arrangement and their impaired interaction in core regions. Due to mutation, the key interfacial interactions between AS160-14-3-3 were changing from Thr642-Asp756, Thr642-Asp757, and Thr642-Lys659 for phosphorylated form, to Ala642-Val681 for mutant. Further, for phosphorylated AS160 the hotspot residues observed were Glu-629, Gln-635, His-641, Lys-653 and Arg-842 which changed to Arg-637, His-641 for mutant. Eventually, the molecular dynamics analysis revealed that local region for phosphorylation site of AS160 is reducing the flexibility, whereas mutation is making the region more flexible. Principal component analysis and Free energy landscape analysis together reveals phosphorylated AS160 is occupying less phase space with more stable landscape when compared to mutant. Our study strongly confers the destabilizing effect of the point mutation at a conserved site providing novel insights right down to the residual level of the conformational dysregulation of AS160 implicating deadly disease. © 2020 Informa UK Limited, trading as Taylor & Francis Group.