This study was conducted to investigate changes in the proximate composition, antioxidant activities, and α-glucosidase inhibitory activity of Opuntia ficus-indica (OFI) cladodes cultivated in Jeju (JJ1, JJ2, JJ3) and Jeonnam (JN1, JN2). The difference in the proximate composition (crude protein, lipid and ash content) of OFI between the two regions was not significant. Ca, Mg and Na were the major mineral components of OFI. The ascorbic acid content of OFI ranged from 57.87 to 143.72 mg/100 g. A 70% ethanol extract was used to investigate the antioxidant content and activity as well as the α-glucosidase inhibitory activity. The total polyphenol and flavonoid contents of OFI were 38.69~55.29 and 3.33~4.03 mg/g, respectively. The antioxidant activities based on the DPPH and ABTS free radical scavenging assays were 45.19~ 61.52% and 39.15~51.96%, respectively, at a concentration of 1 mg/mL. The inhibitory activity of OFI extracts against rat intestinal α-glucosidase was 29.72~45.73% at 1 mg/mL concentration, and JN1 showed the highest α-glucosidase inhibitory activity. This information could be very useful for authentication of Opuntia species with the highest potential as sources of nutritional and therapeutic elements.