Pseudolaric acid B (PAB), a major biologically active component of "TuJinPi" (the root bark of Pseudolarix kaemferi Gordon), exhibited cytotoxicity in many human tumor cell lines. High content analysis (HCA) is a fluorescence microscopy-based automated technology used for quantitative analysis of multiple targets in cells. HCA could yield rich information about the temporal-spatial dynamics of the fluorescence-labeled cell constituents. The mechanism of inhibitory effects of pseudolaric acid B on human breast cancer MCF-7 cells was explored by high content analysis and flow cytometry. As shown by sulforhodamine B assay, PAB inhibited the proliferation of MCF-7 cells in a dose-dependent and time-dependent manner, and the 50% inhibition concentration (IC₅₀) for 72 h was (1.80±0.33) μmol/L. Flow cytometry (propidium iodide staining) showed that, after treatment with PAB for 24 h, the proportion of MCF-7 cells at G2/M phase could increase to about 93%. Flow cytometry (annexin V-FITC and propidium iodide staining) showed that, PAB induced apoptosis of MCF-7 cells. High content analysis showed that: after treatment with PAB for 16 h, the mitotic index of MCF-7 could increase to about 40%, and cyclin B1 was upregulated; PAB caused dose-dependent disassembly of microtubules and inhibited the formation of mitotic bipolar spindles; PAB induced increase of mitochondrial mass; PAB induced grape-like giant nuclei indicating mitotic slippage in MCF-7 cells. These results suggest that PAB inhibits MCF-7 cell proliferation and induces apoptosis, these inhibitory effects may be related to disassembly of microtubules, spindle abnormalities, mitotic arrest and increase of mitochondrial mass.