The tomato genome contains six genes (SlPHO1;1-SlPHO1;6) homologous to AtPHO1. The six proteins have typical characteristics of the plant PHO1 family, such as the three SPX sub domains in the N-terminal portion and one EXS domain in the C-terminal portion. Phylogenetic analysis revealed that the SlPHO1 family is subdivided into three clusters. A pairwise comparison indicated that SlPHO1;1 showed the highest level of sequence identity/similarity (67.39/76.21%) to AtPHO1. SlPHO1;1 deletion mutants induced by CRISPR/Cas9 displayed typical phenotypes of Pi starvation, such as decreased shoot fresh weight (SFW) and increased root fresh weight (RFW), therefore having a greater root-to-shoot (R/S) ratio. Mutants also accumulated more anthocyanin and had more soluble Pi content in the root and less in the shoot. These results indicate that SlPHO1;1 plays an important role in Pi transport in the tomato at seedling stage.