Anaplastic thyroid cancer (ATC) is among the most lethal malignancies. The mitotic kinase PLK1 is overexpressed in the majority of ATCs and PLK1 inhibitors have shown preclinical efficacy. However, they also cause mitotic slippage and endoreduplication, leading to the generation of tetraploid, genetically unstable cell populations. We hypothesized that PI3K activity may facilitate mitotic slippage upon PLK1 inhibition, and thus tested the effect of combining PLK1 and PI3K inhibitors in ATC models, in vitro and in vivo. Treatment with BI6727 and BKM120 resulted in a significant synergistic effect in ATC cells, independent of the levels of AKT activity. Combination of the two drugs enhanced growth suppression at doses for which the single drugs showed no effect, and led to a massive reduction of the tetraploid cells population. Furthermore, combined treatment in PI3Khigh cell lines showed a significant induction of apoptosis. Finally, combined inhibition of PI3K and PLK1 was extremely effective in vivo, in an immunocompetent allograft model of ATC. Our results demonstrate a clear therapeutic potential of combining PLK1 and PI3K inhibitors in anaplastic thyroid tumors. •ATC is one of the most lethal tumor types, with a median survival of less than 6 months from diagnosis.•PLK1 is a mitotic regulator overexpressed in ATC, and the PI3K cascade is constitutively activated in 40% of ATCs.•ATC cells treated with a PLK1 inhibitor often evade cell cycle arrest and death via mitotic slippage and endoreduplication.•PI3K and PLK1 inhibition synergize in vitro, preventing slippage and inducing apoptosis, and potently cooperate in vivo.