Guava (Psidium guajava L., Myrtaceae) leaves have been used as a folk herbal tea to treat diabetes for a long time in Asia and North America. In this study, we isolated polysaccharides from guava leaves (GLP), and evaluated its antioxidant activity in vitro and anti-diabetic effects on diabetic mice induced by streptozotocin combined with high-fat diet. The results indicated that GLP exhibited good DPPH, OH, and ABTS free-radical scavenging abilities, and significantly lowered fasting blood sugar, total cholesterol, total triglycerides, glycated serum protein, creatinine, and malonaldehyde. Meanwhile, it significantly increased the total antioxidant activity and superoxide dismutase (SOD) enzyme activity in diabetic mice, as well as ameliorated the damage of liver, kidney, and pancreas. Thus, polysaccharides from guava leaves could be explored as a potential antioxidant or anti-diabetic agents for functional foods or complementary medicine.