Checkpoint with FHA and Ring Finger (CHFR) is hypothesized to mediate a delay in cell cycle progression early in mitosis in response to microtubule stress, independent of the spindle assembly checkpoint. As a potential regulator of cell cycle progression, CHFR naturally becomes an interesting target for understanding cancer cells. In recent years, there has been increasing evidence supporting the role of CHFR as a tumor suppressor, most of which report loss of expression, occasionally due to promoter hypermethylation, in cancers compared with patient-matched normal tissues. We studied both a panel of breast cancer cell lines as well as primary tissue samples from breast cancer patients to investigate CHFR as a relevant tumor suppressor in breast cancer and to determine whether CHFR expression was associated with clinical and pathologic variables. We report that 41% of cell lines and 36% of patient samples showed low or negative CHFR protein expression or staining. In addition, lack of CHFR detection was associated with increased tumor size and weakly correlated with estrogen receptor–negative tumors from patients. To study the effects of low CHFR expression in vitro, we stably expressed a short hairpin RNA construct targeting CHFR in two lines of immortalized human mammary epithelial cells. Notably, decreased CHFR expression resulted in the acquisition of many phenotypes associated with malignant progression, including accelerated growth rates, higher mitotic index, enhanced invasiveness, increased motility, greater aneuploidy, and amplified colony formation in soft agar, further supporting the role of CHFR as a tumor suppressor in breast cancer. [Cancer Res 2007;67(13):6064–74]