The seasonal appearance and the chemical composition characteristics of cladode of Opuntia humifusa were investigated in this study. The minor (horizontal) and major (vertical) axes, the width, and weight of O. humifusa cladode were compared for its appearance features. Moisture, crude protein, ash, fat, color, dietary fiber, mineral, and amino acids contents were measured for the comparison of their chemical composition characteristics. The cladode of O. humifusa harvested in summer showed higher values of minor and major axis, width, and weight than those harvested in winter. According to the results of Hunter color index, cladode of O. humifusa harvested in summer showed the highest lightness level (Hunter L value). In the meanwhile, cladode of O. humifusa harvested in spring showed the highest Hunter a (redness) and b (yellowness) values. Cladode of O. humifusa harvested in summer had the lowest crude protein and crude fat contents, while it showed the highest crude ash content. The total contents of moisture and dietary fiber were significantly greater than summer-harvested cladode of O. humifusa followed by spring- and winter-harvested cladode of O. humifusa. The major mineral content of all seasonal cladode of O. humifusa was Ca2+. Glutamic acid and aspartic acid were the major amino acids in all seasonal samples. Contrary, the contents of total amino acids and free amino acids were the lowest in summer-harvested cladode of O. humifusa. Taken together, it was concluded that the appearance and chemical quality of cladode of O. humifusa was versatile depending on the harvesting season.