Xylella fastidiosa (Xf) is an insect transmitted, xylem-limited bacterium that causes disease in many economic and ecologically important host plants. A key feature involved in the virulence mechanism of Xf is the formation of biofilm that blocks the flow of water and nutrients from the roots to the leaves. Mineral nutrients such as calcium (Ca) are transported through the xylem vessels. In Xf, calcium was reported to have an important role modulating biofilm formation and twitching motility. Through the study of whole transcriptome analysis and comparative genomics in Xf, a set of genes regulated by calcium was identified. PD0913 was selected for functional analysis since it is also part of a putative genomic island absent in non-virulent strains. Two PD0913-Xf mutants were generated by site-directed mutagenesis and the total, planktonic and biofilm growth was quantified. Compared to the WM1-1 wild type strain, growth of the mutants was reduced under different concentrations of CaCl₂ (2mM, 4mM and 8mM). Biofilm growth was significantly higher under 8mM of Ca, in which WT values were greater than PD0913-Xf mutants. When twitching movement was analyzed, differences between WT and mutant were evident at 4mM Ca, where mutants were impaired in movement. These results suggest that PD0913 gene, so far annotated as a hypothetical protein, is involved in growth and regulation of virulence traits in Xf.