The transition from mitosis to interphase, referred to as mitotic exit, is a critical mitotic process which involves activation and inactivation of multiple mitotic kinases and counteracting protein phosphatases. Loss of mitotic exit checkpoints is a common feature of cancer cells, leading to mitotic dysregulation and confers cancer cells with oncogenic characteristics, such as aberrant proliferation and microtubule-targeting agent (MTA) resistance. Since MTA resistance results from cancer cells prematurely exiting mitosis (mitotic slippage), blocking mitotic exit is believed to be a promising anticancer strategy. Moreover, based on this theory, simultaneous inhibition of mitotic exit and additional cell cycle phases would likely achieve synergistic antitumor effects. In this review, we divide the molecular regulators of mitotic exit into four categories based on their different regulatory functions: 1) the anaphase-promoting complex/cyclosome (APC/C, a ubiquitin ligase), 2) cyclin B, 3) mitotic kinases and phosphatases, 4) kinesins and microtubule-binding proteins. We also review the regulators of mitotic exit and propose prospective anticancer strategies targeting mitotic exit, including their strengths and possible challenges to their use.