The purpose of this study was to investigate the changes in higher fatty acids and related biochemical characteristics of flue-cured tobacco during the process of aging. The concentration of total lipids in flue-cured tobacco decreased gradually during natural aging, but the concentration of individual principal higher fatty acids in flue-cured tobacco changed much differently during aging. Myristic acid (C14:0) increased constantly during the whole aging process. Stearic acid (C18:0) and oleic acid (C18:1) increased at the initial stages of aging and reached maximum contents after one year of aging, but decreased thereafter. Palmitic acid (C16:0), linoleic acid (C18:2), and linolenic acid (C18:3) decreased constantly throughout aging, especially the linolenic acid content which decreased drastically. Furthermore, the ratio of unsaturated fatty acids (C18:1; C18:2; C18:3) and saturated fatty acids (C14:0; C16:0; C18:0) decreased continuously during aging, from 1.00 at the initial stage to 0.43 at the end of aging, when it seemed to be stabilized. The maximum rate of decrease of the ratio was between 12 to 18 mo during aging. The activity of lipoxygenase (LOX) and peroxidation value (POV) initially increased and reached their highest levels after 6 mo of aging, but steadily decreased thereafter. The acid value (AV) and malondialdehyde (MDA) content increased during the first 12 mo of aging, then decreased rapidly after 12 and 18 mo, respectively.