Tomato is a worldwide economic crop, and Fusarium wilt is one of the important tomato diseases which is caused by Fusarium oxysporum f. sp. lycopersici (Fol). The typical symptoms are yellowing and wilting on leaves and browning vascular tissues. Subsequently, it may cause tomato to die or no fruit production. Calcineurin is a heterodimeric phosphatase which can be activated by Ca2+/calmodulin and can dephosphorylate downstream substrates to regulate stress survival, growth, sexual differentiation, and virulence. Calcineurin substrates have been studied in Saccharomyces cerevisiae by phosphoproteomics and quantitative mass spectrometry. To date, there is no research related to proteome analysis of calcineurin downstream substrates in plant pathogens. In our previous study, it revealed that calcineurin is responsible for hyphal growth, conidia formation, chlamydospore formation, and virulence in Fol. In our phosphoproteomics analysis, we compared the protein abundance of wild -type and Δcna1 strains with calcium or non-calcium induced condition. Next, we used Scanprosite and Uniport to predict conserved docking and dephosphorylated sites within those proteins by the abundance more than 2-fold distribution. Finally, we obtained 59 putative calcineurin substrates. According to GO enrichment analysis, these substrates included transcription factors, transmembrane transporters, protein serine/threonine kinase activator, and some uncharacterized proteins. We propose that these putative substrates may play important roles to control physiological function and virulence in Fol.