Reduction of the levels of arsenic in rice shoot is important challenge for agriculture. OsRCS3 is identified as an O-acetylserine(thiol)lyase in present study. OsRCS3 is able to complement an Escherichia coli cys-auxotroph mutant. OsRCS3 is localized to the cytoplasm and the expression of OsRCS3 in roots was up-regulated by arsenic and cadmium treatment. In the presence of As(V), Osrcs3 mutants showed significantly lower levels of Cys, GSH, PC2 in roots compared with wild-type. Knockout of OsRCS3 reduced As accumulation in the roots but increased As accumulation in shoots. We conclude that OsRCS3 is the cytosolic O-acetylserine(thiol)lyase that plays an important role in non-protein thiol synthesis and in restricting As accumulation in rice shoots.