Iron (Fe) homeostasis is critical for plant growth, development and stress response, which is tightly controlled by intricated regulatory networks in which transcription factors (TFs) play a central role. Whilst a series of basic helix-loop-helix (bHLH) TFs have been functionally characterized to contribute to Fe homeostasis, regulatory layers beyond bHLH TFs remain largely unclear. Here, we demonstrate that a SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) TF, LeSPL-CNR, negatively regulates Fe deficiency responses in tomato roots. Fe deficiency rapidly repressed the expression of LeSPL-CNR. However, Fe-deficiency-responses were intensified in two LeSPL-CNR CRISPR/Cas9 gene editing transgenic lines compared with wild-type plants. By comparative transcriptome analyses, we identified 47 Fe-deficiency responsive genes including SlbHLH101 whose expression were negatively regulated by LeSPL-CNR. LeSPL-CNR is localized to nucleus and yeast one-hybrid and luciferase reporter assays revealed that LeSPL-CNR could interact with GTAC motif and BOX4 motif of SlbHLH101 promoter to repress its expression. Moreover, Fe deficiency-induced expression inhibition of LeSPL-CNR is correlated well with SlymiR157 expression induction. Additionally, transgenic tomato overexpressing SlymiR157 displayed improved Fe deficiency responses similar to that of LeSPL-CNR loss-of-function mutants. Altogether, we propose that miR157-LeSPL-CNR module provided an additional pathway that acts upstream of SlbHLH101 to regulate Fe homeostasis in tomato roots.