With the development of molecular biology techniques, intron was known as playing an imperative role in gene’s expression and regulation. Transgenic tobacco IN lines overexpressing InFer1 gene and NT lines overexpressing NtFer1 cDNA gene were obtained, and the exogenous gene expression were confirmed by molecular test. Then for iron content of transgenic tobacco lines and non-transformants as a physiological indicator in status of different iron concentration were measured, and results indicated that the iron content of transgenic tobacco was more than that of non-transformants. In high Fe (II) condition, the NT lines showed higher level in plant height, fresh weight and iron content then that of IN lines, while NT lines showed lower in Malondialdehyde content then IN lines. In soil condition, IN lines showed higher level in plant height, fresh weight, chlorophyll, photosynthesis rate, iron content then that of NT lines. It indicates that intron could play a vital role for improved protective enzyme activity level while reducing reactive oxygen damage and also could help to inhibit the absorption of aborting iron.