The cosmopolitan, bloom-forming diatom, Skeletonema costatum, is a prominent primary producer in coastal oceans, fixing CO2 with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) that is phylogenetically distinct from terrestrial plant RubisCO. RubisCOs are subdivided into groups based on sequence similarity of their large subunits (IA–ID, II, and III). ID is present in several major oceanic primary producers, including diatoms such as S. costatum, coccolithophores, and some dinoflagellates, and differs substantially in amino acid sequence from the well-studied IB enzymes present in most cyanobacteria and in green algae and plants. Despite this sequence divergence, and differences in isotopic discrimination apparent in other RubisCO enzymes, stable carbon isotope compositions of diatoms and other marine phytoplankton are generally interpreted assuming enzymatic isotopic discrimination similar to spinach RubisCO (IB). To interpret phytoplankton δ13C values, S. costatum RubisCO was characterized via sequence analysis, and measurement of its KCO2 and Vmax, and degree of isotopic discrimination. The sequence of this enzyme placed it among other diatom ID RubisCOs. Michaelis-Menten parameters were similar to other ID enzymes (KCO2 = 48.9 ± 2.8 μm; Vmax = 165.1 ± 6.3 nmol min−1 mg−1). However, isotopic discrimination (ε = [12k/13k − 1] × 1000) was low (18.5‰; 17.0–19.9, 95% CI) when compared to IA and IB RubisCOs (22–29‰), though not as low as ID from coccolithophore, Emiliania huxleyi (11.1‰). Variability in ε-values among RubisCOs from primary producers is likely reflected in δ13C values of oceanic biomass. Currently, δ13C variability is ascribed to physical or chemical factors (e.g. illumination, nutrient availability) and physiological responses to these factors (e.g. carbon-concentrating mechanisms). Estimating the importance of these factors from δ13C measurements requires an accurate ε-value, and a mass-balance model using the ε-value for S. costatum RubisCO is presented. Clearly, appropriate ε-values must be included in interpreting δ13C values of environmental samples.