In fields of genetically engineered maize that produce insecticidal Bacillus thuringiensis (Bt) proteins, generalist predators are likely to be exposed to the toxin via prey consumption. For the assessment of risks of Bt proteins to nontarget species, information on Bt protein uptake, fate after feeding, and accumulation after long-term exposure is valuable. One objective of this study was to determine uptake and fate of Bt protein in an arthropod predator. Phylloneta impressa (L. Koch) spiders were fed with a single Diabrotica virgifera virgifera LeConte western corn rootworm beetle or Chrysoperla carnea (Stephens) lacewing that were reared on Cry3Bb1-expressing Bt maize material. The Bt protein concentration measured in spiders 1 d after feeding was 55% of the concentration in beetles and 37% of the concentration in lacewings. Five days after feeding, the Bt protein concentration in P. impressa decreased by ≈90%, indicating rapid excretion, digestion, or both. To investigate the potential accumulation of Cry3Bb1 in spiders, lacewings, and rootworm beetles, concentrations after a short period of feeding (1–8 d) on food containing Bt protein obtained in the present study were compared with published data on long-term continuous feeding (28–64 d). Concentrations of Cry3Bb1 after long-term feeding were similar or lower than those after short-term feeding, which indicates that the Bt protein does not accumulate in the tested arthropods over time.