Xanthomonas citri pv. citri (X. citri pv. citri) is the causal agent of Asiatic citrus canker and infects economically important citrus crops. X. citri pv. citri contains one type VI secretion system (T6SS) required for resistance to predation by the soil amoeba Dictyostelium discoideum and induced by the ECF sigma factor EcfK in the presence of amoeba. In this work, we describe the analysis of T6SS gene expression during interaction with host plants. We show that T6SS genes and the cognate positive regulator ecfK are upregulated during growth in the plant surface (epiphytic) and maintain low expression levels during growth inside plant mesophyll. In addition, expression of the virulence-associated T3SS is also induced during epiphytic growth and shows a temporal induction pattern during growth inside plant leaves. The T6SS is not required for adhesion to leaf surface and biofilm formation during the first stages of plant colonization nor for killing of yeasts cells. Since the phyllosphere is colonized by eukaryotic predators of bacteria, induction of the X. citri pv. citri anti-amoeba T6SS during epiphytic growth suggests the presence of an environmental signal that triggers the resistance phenotype.