To provide a basis for a stable carbon and nitrogen isotope ratio (δ¹³C / δ¹⁵N) analysis to determine the assimilated organic matter in sea cucumber, Holothuria scabra, diet-tissue fractionations were experimentally determined by mono-feeding rearing with diatom. While δ¹⁵N fractionation of the whole body wall (2.4‰) was similar to the commonly accepted value (2.6 - 4‰), δ¹³C fractionation of the body wall (4.2‰) showed considerable discrepancy with the commonly accepted value (0 - 1‰) due to the high content (35% dry wt/wt) of calcareous spicules (CaCO₃) in the body wall, which had significantly higher δ¹³C (-8.6‰) than the organic fractions. Computational elimination of spicules based upon spicule content and spicule δ¹³C reduced the δ¹³C fractionation of the body wall to 1.5‰, close to the common value. δ¹³C fractionation after spicule removal by acid decarbonation and subsequent rinsing (3.2‰) did not agree with the common value, and δ¹⁵N fractionation was significantly elevated by decarbonation. δ¹⁵N and δ¹³C fractionations of the intestine (1.5 and 2.2‰, respectively) did not agree with the common values. Since δ¹³C and δ¹⁵N of the feces did not differ significantly from those of the diet, feces may be used to determine ingested organic matter in the wild.