The present study investigated the antidiabetic potential of protein isolates from Hawaijar (HPI), a popular fermented soybean food of North-East India. Treatment with HPI significantly upregulated glucose uptake, glucose utilization, glucose-6-phosphate, and stimulated PI3K/AKT/GLUT4 pathway in high-glucose (HG)-treated myotubes. Signal silencing studies demonstrated that knockdown of insulin-dependent signaling molecule (IR) but not insulin-independent signaling molecule (AMPK) significantly inhibited HPI-induced activation of PI3K/AKT/GLUT4 pathway and glucose uptake in HG-treated myotubes. SDS-PAGE and immunoblotting analyses of HPI showed the reduction and/or absence of various subunits of 7S and 11S globulin protein and appearance of new proteins compared to respective non-fermented soy protein isolates. Using various chromatographic techniques, the present study further isolated a single protein (ISP, ~24 kDa) from HPI as one of the bioactive principles with promising glucose utilization potential via stimulating PI3K/AKT/GLUT4 pathway in HG-treated cells. ISP treatment along with insulin significantly stimulated PI3K/AKT/GLUT4 pathway and glucose uptake compared to either insulin or ISP alone treated cells against HG exposure suggesting the insulin sensitizing effect of ISP. Furthermore, ISP supplementation significantly reduced metabolic markers linked with diabetes in high-fructose high-fat diet-fed animal model of type 2 diabetes. This study demonstrated a novel molecular mechanism underlying the promising antidiabetic potential of HPI.