Mitochondrial complex I (CI) as a critical multifunctional respiratory complex of electron transport chain (ETC) in mitochondrial oxidative phosphorylation has been identified as vital and essence in ATP production, biosynthesis and redox balance. Recent progress in targeting CI has provided both insight and inspiration for oncotherapy, highlighting that the development of CI-targeting inhibitors is a promising therapeutic approach to fight cancer. Natural products possessing of ample scaffold diversity and structural complexity are the majority source of CI inhibitors, although low specificity and safety hinder their extensive application. Along with the gradual deepening in understanding of CI structure and function, significant progress has been achieved in exploiting novel and selective small molecules targeting CI. Among them, IACS-010759 had been approved by FDA for phase I trial in advanced cancers. Moreover, drug repurposing represents an effective and prospective strategy for CI inhibitor discovery. In this review, we mainly elaborate the biological function of CI in tumor progression, summarize the CI inhibitors reported in recent years and discuss the further perspectives for CI inhibitor application, expecting this work may provide insights into innovative discovery of CI-targeting drugs for cancer treatment.