Cyanate (CNO−) has been produced in the environment through either natural or anthropogenic sources. However, due to industrialization, it has been led more over-loads. In this study, interaction of CNO− uptake by rice seedlings with nitrate assimilation was investigated using gene expression analysis after an acute phytotoxicity assay. Our results showed that CNO− exposure caused inhibition on relative growth rates of plants. CNO− analysis demonstrated that rice seedlings had higher potential for CNO− uptake and the removal rates showed a zero-order kinetic. PCR analysis exposed that OsCYN transcript was not significantly induced by CNO− treatments in rice tissues and CNO− exposure also repressed gene expression of the collaborative enzyme carbonic anhydrase (CA), suggesting that assimilation of CNO− initiated by the enzyme cyanase (CYN) in rice seedlings was an enzyme-limitation reaction. Gene expression of other enzymes involved in nitrate metabolism was tissue-specific under CNO− exposure, suggesting that rice seedlings were able to trigger its intrinsic regulative and responsive mechanisms to cope up with uneven N conditions. Significant upregulation of three OsGDH isogenes, except for OsGDH1 in roots, was detected in both rice materials with enhancing CNO− concentrations, suggesting that GDH may play a primary role to maintain the balance of C and N in plants under CNO− exposure. In conclusion, because the innate pool of CYN activity was non-sufficient to degrade exogenous CNO− by rice seedlings, CNO-derived ammonium only can serve as a supporting N source to support growth of rice seedling under non-effective doses of CNO− exposure.