Successful responses to radioiodine treatment in differentiated thyroid cancer ultimately depend on uptake via the sodium-iodide symporter (NIS). However, many tumors exhibit NIS dysregulation, resulting in a poorer prognosis. Since breast cancer can also overexpress NIS, albeit of limited function, radioiodine treatment may be a promising treatment option. Our previous data show that overexpression of the pituitary tumor-transforming gene-binding factor (PBF) is partially responsible for the reduced function of NIS in thyroid and breast cancer. PBF interaction with NIS leads to reduced NIS plasma membrane localization, decreasing functionality. NIS binding requires a C-terminal PBF tyrosine residue 174 (Y174) to be phosphorylated by the protein kinase Src and hence radioiodine uptake can be modified by Src overexpression and inhibition. To address the mechanistic interactions between NIS, PBF and Src we used CRISPR/Cas9 to knock PBF out in Nthy-ori 3-1 normal thyroid and TPC1 thyroid cancer cells, as well as in MDA-MB-231 and MCF7 breast cancer cell lines. Endonuclease screening, Western blotting and DNA sequencing identified successful PBF knock out (PBF-KO) with at least two different guide RNAs (gRNA). Src overexpression in parental TPC1, MDA-MB-231 and MCF7 cells expressing exogenous NIS significantly repressed radioiodine uptake. In contrast, radioiodine uptake was not altered with Src overexpression in the PBF-KO cell lines. Interestingly Src overexpression had no effect on radioiodine uptake in the Nthy-ori cells. In an alternative approach we previously targeted Src myristoylation and demonstrated increased radioiodine uptake with an N-myristoyltransferase inhibitor. We now show that NMTi significantly induced radioiodine uptake in TPC1 and Nthy-ori parental cells but had no effect in PBF-KO cells. Thus, it is likely that the ability of Src to repress NIS function is dependent on PBF. So, Src inhibitors may contribute to the restoration of radioiodine uptake in thyroid cancer and the utilization of radioiodine in breast tumors.