The stomatal characteristics, length and dry weight as well as stable carbon isotope composition (d13C) of needles and tree rings of Qinghai spruce (Picea crassifolia) at different altitudes in the Qilian mountains were investigated. The results showed that stomatal density, distribution pattern on epidermis, and length and dry weight of needles all increased with altitude below 3,000m. In contrast, these parameters all decreased with increasing altitude above 3,000m. Furthermore an assay of tree rings showed that near 3,000m in altitude was the optimum zone for growth and development of Qinghai spruce. Although atmospheric CO2 concentration influences stomatal density, the effects of many environmental factors, such as temperature, light and rainfall, could obscure the effects of changes in atmospheric CO2 concentration on stomatal density at altitudes higher than the optimum for growth. The correlation of stomatal density with atmospheric CO2 concentration and d13C of Qinghai spruce needles was significant below 3,000m, but was insignificant above 3,000m altitude. The d13C value of needles, however, was negatively correlated with atmospheric CO2 concentrations. Therefore, the influence of altitude should be considered in research on plant physiological ecology and the relationship of stomatal density with d13C value or atmospheric CO2 concentration.