The ratio of intercellular to ambient CO2 concentrations (ci/ca) plays a key role in ecophysiology, micrometeorology, and global climatic change. However, systematic investigation on ci/ca variation and its determinants are rare. Here, the ci/ca was derived from measuring ecosystem fluxes in an even-aged monoculture of rubber trees (Hevea brasiliensis). We tested whether ci/ca is constant across environmental gradients and if not, which dominant factors control ci/ca variations. Evidence indicates that ci/ca is not a constant. The ci/ca exhibits a clear “V”-shaped diurnal pattern and varies across the environmental gradient. Water vapor pressure deficit (D) is the dominant factor controls over the ci/ca variations. ci/ca consistently decreases with increasing D. ci/ca decreases with square root of D as predicted by the optimal stomatal model. The D-driving single-variable model could simulate ci/ca as well as that of sophisticated model. Many variables function on longer timescales than a daily cycle, such as soil water content, could improve ci/ca model prediction ability. Ecosystem flux can be effectively used to calculate ci/ca and use it to better understand various natural cycles.