Tea plant (Camellia sinensis (L.) O. kuntze) is known to be a fluoride (F) and aluminum (Al3+) hyper-accumulator. Previous study showed that pre-treatment of Al3+ caused a significant increase of F accumulation in tea plants. However, less is known about the intricate network of Al3+ promoted F accumulation in tea plants. In this study, the involvement of endogenous Ca2+ and CaM in Al3+ pretreatment-promoted F accumulation in tea plants was investigated. Our results showed that Al3+ induced the inverse change of intracellular Ca2+ fluorescence intensity and stimulated Ca2+ trans-membrane transport in the mature zone of tea root. Also, a link between internal Ca2+ and CaM was found in tea roots under the presence of Al3+. In order to investigate whether Ca2+ and CaM were related to F accumulation promoted by Al3+ pretreatment, Ca2+ chelator EGTA and CaM antagonists CPZ and TFP were used. EGTA, CPZ, and TFP pretreatment inhibited Al3+-induced increase of Ca2+ fluorescence intensity and CaM content in tea roots, and also significantly reduced Al3+-promoted F accumulation in tea plants. Taken together, our results suggested that the endogenous Ca2+ and CaM are involved in Al3+ pretreatment-promoted F accumulation in tea roots.