Abstract Objectives This study aimed to explore the pharmacological properties of pinoresinol-4-O-β-D-glucopyranoside (PG), isolated from prunes. Methods In-vitro antioxidant activity was assessed using ferric reducing antioxidant power (FRAP) and 2,2'-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) assays. In-vivo hepatoprotective activity was evaluated using CCl4-induced hepatotoxicity mouse model. The antihyperglycaemic activity was determined in vitro using α-glucosidase and α-amylase inhibiting activity and in vivo using streptozotocin-treated model. Molecular modelling was done on α-amylase, α-glucosidase, aldose reductase and peroxisome proliferator-activated receptor gamma. Key findings Pinoresinol-4-O-β-D-glucopyranoside showed promising antioxidant activity in FRAP and ABTS assays with total antioxidant capacity equal 418.47 and 1091.3 µmol/g in terms of ascorbic acid, respectively. PG (50 mg/kg b.w.) exhibited a hepatoprotective activity in vivo as it lowered AST and ALT levels. PG showed a potent in-vitro antihyperglycaemic activity as it inhibited α-glucosidase with an IC50 value of 48.13 μg/ml. PG caused a prominent decline in serum glucose level by 37.83% in streptozotocin-treated mice with promising elevation in insulin level of 25.37%. Oxidative stress markers were reduced by PG, and it showed a high fitting on α-amylase and α-glucosidase active sites. Conclusions Pinoresinol-4-O-β-D-glucopyranoside is a natural entity combating oxidative stress, hepatic damage and diabetes.