We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu-uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high- and low-affinity Cu-transport systems. The half-saturation constants (Km) and the maximum Cu-uptake rates (Vmax) of the high-affinity Cu-transport systems (∼7–350 nM and 1.5–17 zmol · μm−2 · h−1, respectively) were significantly lower than those of the low-affinity systems (>800 nM and 30–250 zmol · μm−2 · h−1, respectively). The two Cu-transport systems were controlled differently by low Fe and/or Cu. The high-affinity Cu-transport system of both diatoms was down-regulated under Fe limitation. Under optimal-Fe and low-Cu growth conditions, the Km of the high-affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low-affinity Cu-transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu-transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.