Tenderization of beef using proteolytic enzymes often leads to high water loss due to harsh structural disruption. This study first verified the enzymatic deamidation (protein-glutaminase, PG) in softening grass-fed steak texture without exacerbating the cooking loss. PG deamidation at the deamidation degree (DD) of 5% (d5) or 7% (d7) significantly increased the tenderness and softened the texture whereas they negligibly affected the cooking loss. The muscle bundles were loosed while fibers were thickened probably as the result of charge induced myofibril dissociation (increased myofibril fraction index) and intermolecular repulsion. Compared to papain, the effect of PG deamidation on the water mobility was minor, though it was still seen a slight conversion of bound water into immobilized water in high DD steaks (d5 and d7). PG deamidation hardly hydrolyze individual proteins but partially altered the tertiary structure of muscle proteins, resulting in a slight decrease in denaturation enthalpies of muscle proteins including myosin, collagen and sarcoplasmic protein. These results demonstrated that PG deamidation appeared to be a novel and promising strategy in meat tenderization.