Olive shoots have a hard-rooting nature that causes significant problems associated with olive micro-propagation under in vitro conditions. Several factors may contribute to alleviating this problem, in cluding zinc and its derivatives. The current research involved the application of zinc oxide nanoparticles and zinc sulfate on olive explants ‘Caillette’, ‘Beldi’ and ‘Dezfoli’ cultivars. FT-IR spectroscopy, UV-Vis spectroscopy, FESEM, EDS, and DLS techniques characterized the zinc oxide nanoparticles. Then, micro cuttings containing two buds were cultured on a half-strength MS medium containing 1.5 mg L-1 of BAP hormone and 1 mg L-1 of GA3 hormone. The treatments led to olive shoots, 3 cm in length that were transferred to a 1/2 MS medium containing 3 mg L-1 of IBA. The olive shoots were treated with two types of zinc compounds, including zinc sulfate (0.0, 2.43, and 4.86 mg L-1) and zinc oxide nanoparticles (0.0, 2.43, 4.86, and 7.29 mg L-1). The results showed that zinc oxide nanoparticles induced callus growth in response to all concentrations but prevented root growth. Alternatively, zinc sulfate at all concentrations induced root and callus growth, although to a smaller extent than nanoparticles. The ‘Caillette’ cultivar had the highest rooting percentage and heaviest fresh and dry root weight. The ‘Beldi’ cultivar had the lowest of these values. Several factors potentially contributed to these results, such as hormonal (auxin biosynthesis), biochemical (enzyme and other proteins), and molecular factors (bZIP TFs), which changed by zinc application. A complementary bioinformatics study was conducted as well.