In the present study, the arsenic (As) biokinetics and bioavailability in two deposit-feeding invertebrates (clams Gafrarium tumidum and polychaetes Nereis succinea) were quantified. Radiotracer techniques were applied to measure the dissolved uptake rate, dietary assimilation efficiency and efflux of As by the clams and polychaetes. Simultaneously, arsenic species analysis was conducted to examine the As biotransformation following dietary uptake. The radiotracer results showed that the uptake rate constant and efflux rate constant were 0.068L/g/d and 0.07d−1, and 0.173L/g/d and 0.09d−1, in the clams and polychaetes, respectively. Sediments labeled for different times (1.5–60 d) with different inorganic/organic As percentages led to diverse assimilation efficiencies of As (35.1–56.1% in the clams, and 51.6–72.6% in the polychaetes). Modeling calculations showed that sediment was a significant source for As bioaccumulation in the two deposit-feeders. After feeding on the spiked sediments, inorganic As (75.6%) was initially the predominant form, but arsenobetaine (AsB) became the predominant compound (>90%) in the clams and polychaetes during depuration, suggesting biotransformation of inorganic As. Combined with the biokinetics and biotransformation measurements, we showed that AsB was more efficiently assimilated and tended to be accumulated, whereas As(III) was less efficiently assimilated and more rapidly eliminated by the two invertebrates. This study demonstrated that As speciation in the sediments as a significant source for As bioaccumulation caused different bioavailability in deposit-feeding clams and polychaetes.