Purpose <p>Differentiated thyroid cancer (DTC) typically has a favourable prognosis, while advanced forms of these tumours exhibit aggressive behaviour, leading to decreased survival. Lenvatinib has demonstrated effectiveness in managing advanced DTC. However, its long-term efficacy is compromised by resistance mechanisms, which remain unexplored, limiting its clinical utility.</p> Methods <p>In vitro studies were conducted using three advanced DTC cell lines of the papillary subtype: K1 (<i>BRAF</i> p.V600E), BCPAP (<i>BRAF</i> p.V600E) and TPC-1 (<i>RET/PTC1</i>). IC₅₀ for Lenvatinib and Gefitinib were defined, and their effects on cell viability, colony formation, gene expression, and pathway activation were assessed individually and in combination. Comparative genomic hybridisation was performed to uncover intrinsic resistance mechanisms.</p> Results <p>Lenvatinib IC₅₀ was higher in K1 and BCPAP than in TPC-1, indicating greater TPC-1 susceptibility, as confirmed by viability assays. Gefitinib showed similar IC₅₀ values across all cell lines. The Lenvatinib plus Gefitinib combination (Combo) significantly reduced K1 and BCPAP viability when comparing with monotherapies, with no significant effects in TPC-1. Clonogenic assays mirrored these results and revealed higher recovery in K1 and BCPAP after Combo withdrawal. Combo treatment increased EGFR expression and induced ERK1/2 and AKT phosphorylation’s dysregulation in all cell lines.</p> Conclusions <p>This study showed that <i>RET/PTC1</i> cells are more responsive to Lenvatinib than <i>BRAF</i>-mutated cells, and that EGFR targeting with Gefitinib enhanced Lenvatinib’s inhibitory effect in <i>BRAF</i>-mutated cells. Dysregulation of EGFR and Lenvatinib target gene’s expression, as well as of MAPK and PI3K signalling may indicate short-term adaptive resistance. These findings provide insight into Lenvatinib resistance mechanisms in thyroid cancer and highlight the need for further research to overcome refractoriness.</p>