Numerous studies have identified Thymoquinone (TQ) as a potential antidiabetic agent. However, its low solubility in water hampers bioavailability. This study focuses on the impact of PEGylation on the antidiabetic properties of TQ. We found that encapsulating TQ with PEG4000 was highly effective, achieving an encapsulation efficiency of over 99 %. The stability of this formulation, TQ-PEG4000-NP, was assessed by monitoring the TQ release at different pH levels. We evaluated cytotoxicity in two cell lines, EMT6 and NIT-1 (β-cells). Free TQ showed moderate cytotoxicity towards NIT-1 cells but was non-toxic to EMT6 cells. Remarkably, TQ-PEG4000-NP significantly enhanced the viability of the NIT-1 cells. Further studies assessed insulin secretion in isolated Langerhans islets and NIT-1 cells. Both free and PEGylated TQ markedly improved insulin secretion (75 μl U.ml-1 in control and 140 μl U.ml-1 in islets treated with TQ and TQ-PEG4000-NP). In addition, PEGylation substantially increased the viability of STZ-treated β-cells. The beneficial effects of both TQ and TQ-PEG4000-NP are linked to their influence on oxidative stress markers, such as GSH, MDA, and SOD. This study highlights the effectiveness of thymoquinone as an anti-diabetic agent, emphasizing its protective and regenerative effects on β-cells and Langerhans islets, and its ability to enhance insulin secretion. Encapsulation with PEG4000 mitigates TQ's cytotoxicity of TQ to β-cells, bolsters its cytoprotective and regenerative properties, and maintains its insulin-stimulating capability.