Taxanes, diterpenoids produced by the genus Taxus L., are used to treat a range of cancers, especially paclitaxel (Taxol®), whose therapeutic use has already been confirmed by the U.S. Food and Drug Administration. Taxus baccata L. cell culture is a promising commercial technique for the production of taxanes. In the present study, the effect of gold nanoparticles (AuNPs) (0, 1, 2, and 4 µg/mL) at various exposure time (8, 24, 48, and 72 h) on cell morphological changes, biochemical traits, and expression level of taxanes biosynthesis-related genes (txs, t13αoh, bapt, and dbndpt), and taxanes content in a T. baccata cell culture were studied. The ultra-structural changes of the treated cells were observed with increasing the level and exposure time of AuNPs. The maximum intracellular total phenol content was determined 8 h following treatment of 4 µg/mL AuNPs. The highest average of total intracellular (1.21 mg/L) and extracellular (1.04 mg/L) content of taxanes were obtained at 2 µg/mL AuNPs after 24 h, exhibiting a total increase of 1.4-fold higher than untreated cells. A 137-fold increase in the expression level of bapt at 2 µg/mL AuNPs, 8 h after treatment resulted in a 7.5- and 2.5-fold improvement in accumulation of 10-deacetyl taxol and taxol, respectively. Treated T. baccata cells with 2 µg/ mL AuNPs after 8 h showed a 49-fold increase in the expression level of txs, leading to a 1.5-fold increase in taxanes 24 h later.