The effectiveness of in situ stabilization in the long-term As-contaminated soil was assessed. In situ stabilization of As was conducted through a Fe-based sorbent amendment. Chemical extractability of As was first determined by solubility/bioavailability research consortium extraction method and any change in human health risk through oral ingestion was characterized. Also, nonspecifically bound As in soil was determined by five-step sequential extraction. The results indicate that such extractable fractions of As decreased, and consequently risk through oral ingestion decreased probably due to hematite transformed from both the goethite in the original soil and the Fe-based sorbent, which was identified through the X-ray absorption spectroscopy. In ecotoxicity test with Hondeum vulgare, root and shoot elongation and germination rate decreased which was contrary to the chemical extraction data. Such increase in As toxicity is because of increased exchangeable Ca2+ concentration causing As accumulation in the membrane surface of H. vulgare. Also, adsorption of phosphorus onto the Fe-based sorbent decreased available phosphorus concentration causing phosphorus deficiency for growth. Our results demonstrate that the effectiveness of in situ stabilization should be evaluated by means of both chemical extractability and biological response, as chemical analysis alone may not be sufficient to assess the ecotoxicity.