Oxalate is widely distributed in the plant kingdom. While excess oxalate in food crops is detrimental to animal and human health, it may play various functional roles in plants, particularly for coping with environmental stresses. Understanding its biosynthetic mechanism in plants, therefore, becomes increasingly important both theoretically and practically. However, it is still a matter of debate as to what precursor and pathway are ultimately used for oxalate biosynthesis in plants. In this study, both physiological and molecular approaches were applied to address these questions. First, it was observed that when glycolate or glyoxylate was fed into detached leaves, both organic acids were equally effective in stimulating oxalate accumulation. In addition, the stimulation could be completely inhibited by cysteine, a glyoxylate scavenger that forms cysteine-glyoxylate adducts. To verify the role of glyoxylate further, various transgenic plants were generated, in which several genes involved in glyoxylate metabolism [i.e. SGAT (serine-glyoxylate aminotransferase), GGAT (glutamate-glyoxylate aminotransferase), HPR (hydroxypyruvate reductase), ICL (isocitrate lyase)], were transcriptionally regulated through RNAi or over-expression. Analyses on these transgenic plants consistently revealed that glyoxylate acted as an efficient precursor for oxalate biosynthesis in rice. Unexpectedly, it was found that oxalate accumulation was not correlated with photorespiration, even though this pathway is known to be a major source of glyoxylate. Further, when GLDH (L-galactono-1,4-lactone dehydrogenase), a key enzyme gene for ascorbate biosynthesis, was down-regulated, the oxalate abundance remained constant, despite ascorbate having been largely reduced as expected in these transgenic plants. Taken together, our results strongly suggest that glyoxylate rather than ascorbate is an efficient precursor for oxalate biosynthesis, and that oxalate accumulation and regulation do not necessarily depend on photorespiration, possibly due to the occurrence of the anaplerotic reaction that may compensate for glyoxylate formation in rice.