Telomeres adopt a strand-invaded lariat configuration, or T-loop, which physically conceals the chromosome termini from the DNA damage response. Prolonged mitotic arrest induces dissociation of the telomere binding protein TRF2, T-loop opening, and subsequent deprotection of the chromosome end. This mitotic arrest-dependent telomere deprotection activates a terminal tumour suppressive checkpoint that triggers cell death in response to mitotic chemotherapies through undefined mechanisms. We therefore explored the regulation of mitotic deprotection aiming to identify the key molecular regulators. Application of proximity biotinylation through the ascorbic acid peroxidase APEX2 fused to the telomere-binding protein TRF1, identified that components of the BTR helicase, and Aurora B kinase complexes localise to the telomere during mitotic arrest. Both complexes are recruited by a domain of TRF1, modified by phosphorylation of Ser354, and Thr358. Knock-down, inactivating mutation or chemical inhibition of either complex impairs deprotection. Phospho-mimetic mutations of consensus Aurora B sites Ser62/65 on the N-terminus of TRF2 strengthen deprotection and may impair T-loop binding. We propose a model whereby coordinated phosphorylation of TRF1 during mitotic arrest recruits both the BTR and Aurora B kinase complexes to the telomere. Once localised, Aurora B weakens the interaction between TRF2 and the telomere, allowing the BTR helicase complex to dissolve the T-loop structure. Decoupling this network can accelerate deprotection, and cell death. This investigation provides evidence that mitotic deprotection could utilised pharmacologically to enhance the efficacy of widely used mitotic chemotherapies.