This study highlights the roles of different N sources (N starvation [−N], nitrate [+NO 3 − ], and ammonium [+NH 4 + ]) in specific amino acids involved in rice proline (Pro) metabolism under hexavalent chromium [Cr(VI)] stress. In treatment with Cr(VI) − N, the content of glutamate (Glu) in shoots was comparable to that of Pro, while the content of Glu in the roots was significantly higher than that of Pro. In case of treatment with Cr(VI) + NO 3 − /NH 4 + , the content of Glu in both tissues was significantly higher than that of Pro, suggesting that the synthesis of γ-aminobutyric acid and glutathione was most likely involved. Pro synthesis in rice is mainly derived from the Glu pathway rather than the ornithine (Orn) pathway, which is strongly affected by different N sources. The genetic regulation network of Pro metabolism demonstrated a good signal for describing amino acid balance in rice under Cr(VI) stress.