This study aims to explain the mechanism of action of allelochemical effect of Chenopodium ambrosioides L. volatile oil on mitochondrial characteristics of receptor plants. This study chose maize (Zea mays L.), which is widely grown in C. ambrosioides invaded field as the receptor plant, and investigated the allelochemical effect of C. ambrosioides volatile oil and its two main components (α-terpinene and cymene) on maize root tip cells using a suspended gas method and TdT-mediated dUTP Nick-End Labeling (TUNEL) assays. The allelochemical effect on the relative expression of the apoptosis-associated type II Metacaspase gene ZmMCII was measured by quantitative real time polymerase chain reaction (qRT-PCR). The allelochemical effect on mitochondrial characteristics was analyzed by staining assays. The results showed that applications of C. ambrosioides volatile oil, α-terpinene, and cymene on maize root tip cells caused membrane lipid peroxidation, increased mitochondrial H2O2 and MDA contents, decreased mitochondrial membrane potential, released mitochondrial Cyt c and Ca2+ into the cytoplasm, and increased cytosolic Ca2+ level and expression of the type II Metacaspase gene ZmMCII. These apoptotic effects were time-dependent. It suggested that C. ambrosioides volatile allelochemicals induced accumulation of reactive oxygen species (ROS) and triggered mitochondrion-mediated Ca2+- and Caspase-dependent apoptosis signaling pathways in receptor plant cells.