The Na+/I- symporter (NIS) mediated iodide uptake activity is the basis for targeted radioiodide ablation of thyroid cancers. While it has been shown that NIS protein is phosphorylated, neither the in vivo phosphorylation sites nor their functional significance have been reported. In this study, S43, T49, S227, T577, and S581 were identified as in vivo NIS phosphorylation sites by mass spectrometry. Kinetic analysis of NIS mutants in the corresponding phosphorylated amino acid residue indicates that the velocity of iodide transport of NIS is modulated by the phosphorylation status of S43 and S581. We also found that the phosphorylation status of T577 may be important for NIS protein stability, and that the phosphorylation status of S227 is functionally silent. T49 appears to be critical for proper local structure/conformation of NIS since mutation of T49 to alanine, aspartic acid, or serine, all resulted in reduced NIS activity without alterations in total or cell surface NIS protein levels. Taken together, we showed that NIS protein levels and functional activity could be modulated by phosphorylation through distinct mechanisms.