We present here the effects of ambient ozone (O3)-induced decline in carbon availability, accelerated foliar senescence, and a decrease in aboveground biomass accumulation in the Aleppo pine (Pinus halepensis Mill.). Aleppo pine seedlings were continuously exposed in open-top chambers for 39 months to three different types of O3 treatments, which are as follows: charcoal-filtered air, nonfiltered air (NFA), and nonfiltered air supplemented with 40 ppb O3 (NFA+). Stable carbon isotope discrimination (Δ) and derived time-integrated ci/ca ratios were reduced after an accumulated ozone exposure over a threshold of 40 ppb (AOT40) value from April to September of around 20,000 ppb·h. An AOT40 of above 67,000 ppb·h induced reductions in ribulose-1, 5-biphosphate carboxylase/oxygenase activity, aboveground C and needle N and K concentrations, the C/N ratio, Ca concentrations in twigs under 3 mm, and the aerial biomass, as well as increases in needle P concentrations and phosphoenolpyruvate carboxylase (PEPC) activity and the N and K concentrations in twigs under 3 mm. Macronutrients losses, the limitations placed on carbon uptake, and increases in catabolic processes may be the causes of carbon gain diminution in leaves which was reflected as a reduction in aboveground biomass at tree level. Stimulation of PEPC activity, the consequent decreased Δ, and compensation processes in nutrient distribution may increase O3 tolerance and might be interpreted as part of Aleppo pine acclimation response to O3.