Alternative splicing provides additional genomic complexity by producing multiple mRNAs and protein variants from any given gene. Splice variants have been identified in a large variety of cancer genes, suggesting that widespread aberrant and alternative splicing may be a consequence or even a cause of cancer. Human ortholog of mammalian enabled (hMena), a family of enabled/vasodilator-stimulated phosphoproteins (Ena/VASP), is an actin regulatory protein involved in the regulation of cell motility. hMena has been shown to have several splice variants, including the hMena(INV) isoform, expressed in invasive cancer cells, and the epithelial-specific isoform, hMena(11a). We assessed the relative mRNA expression of hMena splice variants in 50 cases of invasive ductal breast carcinoma of no special type (IDC-NST) and 45 cases of ductal breast carcinoma in situ (DCIS) with special reference to non-neoplastic breast epithelial tissues. The samples were dissected from their respective regions by laser microdissection. Our results confirmed previous reports that hMena(INV) expression is augmented during tumor progression, while hMena(11a) is downregulated. Furthermore, simultaneous expression of hMena(11a) and hMena(INV) was found only in malignant lesions, while their expression was hardly detected in normal breast tissue and benign proliferative breast lesions. These results indicate that the higher relative expression of hMena(11a) compared with hMena(INV) may predict malignant transformation in breast epithelial cells, and, furthermore, a reversal of expression of hMena(11a) and hMena(INV) may dictate the state of cancer progression. Here, we demonstrate that determination of hMena(11a) and hMena(INV) expression could be a useful biomarker for predicting malignant behavior in breast epithelial lesions, and show that their relative expression is linked to adverse prognostic factors. Although the biological activity of the majority of alternatively spliced isoforms and their contribution to cancer biology has yet to be determined, their elucidation will have a large impact on therapeutic strategies for cancer.