Seabuckthorn (Hippophae rhamnoides L.) is a small fruit tree in the family Elaeagnaceae, which is distributed in Asia and Europe. Because the fruit of this species are rich in vitamins and unsaturated fatty acids, seabuckthorn has potential as a food and medicinal crop. Production of seabuckthorn fruit has begun worldwide. We compared the nutritional components of fruits of two seabuckthorn subspecies, H. rhamnoides ssp. mongolica and ssp. rhamnoides, that are grown in Japan. The sugars and acids that are important in fruit flavor were measured in both subspecies. The glucose content was the highest among the sugars, suggesting that the flavor of the fruit might be improved by enhancing the accumulation of sweeter sugars such as fructose and sucrose. The fruits of ssp. mongolica contained higher concentrations of sugars and a lower acid content than ssp. rhamnoides, indicating that ssp. mongolica fruits have better flavor. Seabuckthorn fruits also contained high levels of ascorbic acid. The ascorbic acid content in fruits of ssp. mongolica was lower than in fruits of ssp. rhamnoides. Therefore, the metabolic pathway for the biosynthesis of ascorbic acid in this species was investigated with the goal of improving the ascorbic acid content. Galacturonate reductase (GalUR), L-galactose dehydrogenase (L-GDH), and myo-inositol oxygenase (MIOX) are potential key enzymes in the galacturonate, mannose/L-galactose, and myo-inositol pathways for ascorbic acid synthesis, respectively. The activities of each of these enzymes were detected in fruits, with the MIOX activity much higher than the GalUR or L-GDH activities. The GalUR activity increased during ripening, as observed in strawberry, whereas the ascorbic acid decreased, suggesting that there is no relationship between the biosynthesis of ascorbic acid and the degradation of cell-wall pectin. It is possible that the substrate for the biosynthesis of ascorbic acid in seabuckthorn fruit is supplied from MIOX and/or L-GDH.