The effect of pruning (cut-back at 5cm height) in comparison to no pruning (plants with central leader) was studied at eco-physiological and anatomical levels in olive trees. Photosynthetic rate, stomatal conductance, leaf water potential, leaf osmotic potential, turgor potential, relative water content, and different anatomical features were reported. The results indicate that, non-pruned plants presented higher value of photosynthetic rate and stomatal conductance compared to the pruned plants. In addition, they maintain higher turgor potential for the same values of relative water content (RWC). At anatomical level, non-pruned plants exhibited higher relative volume of spongy cells, spongy thickness and significantly higher number of vascular bundles per leaf section compared to the pruned plants. Palisade parenchyma thicknesses as well as non-glandular hair density (No./mm2) were lower in pruned plants. Possible effect of pruning on juvenility is discussed.