The DNA damage response (DDR) mediates the recognition of different types of DNA damage, cell cycle checkpoint, and DNA repair pathways to maintain genomic stability. In cancer cells, the defects or mutations in the genes of the DDR and associated pathways induce genomic instability. Therefore, targeting the DDR pathway has great attention for anticancer drug development. DNA damages are recognized by the phosphoinositide 3-kinase-related kinase (PIKK) family, including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PKcs). Among them, the targeting of ATR has significant therapeutic potential in treating cancer with defective DDR signaling. We reviewed the essential role of ATR in the DDR pathway, especially with cancer development, and comprehensively focused on the preclinical investigations to reveal the therapeutic efficacy of ATR inhibitors in cancer therapy. Additionally, we describe the progress and current status of ATR inhibitors in cancer treatment with preclinical and clinical studies.