Human mena (hMENA), a member of the actin cytoskeleton regulators Ena/VASP, is overexpressed in high-risk preneoplastic lesions and in primary breast tumors and has been identified as playing a role in invasiveness and poor prognosis in breast cancers that express HER2. Here we identify a unique isoform, hMENAΔv6, derived from the hMENA alternative splicing program. In an isogenic model of human breast cancer progression, we show that hMENA(11a) is expressed in premalignant cells, whereas hMENAΔv6 expression is restricted to invasive cancer cells. "Reversion" of the malignant phenotype leads to concurrent down-regulation of all hMENA isoforms. In breast cancer cell lines, isoform-specific hMENA overexpression or knockdown revealed that in the absence of hMENA(11a), overexpression of hMENAΔv6 increased cell invasion, whereas overexpression of hMENA(11a) reduced the migratory and invasive ability of these cells. hMENA(11a) splicing was shown to be dependent on the epithelial regulator of splicing 1 (ESRP1), and forced expression of ESRP1 in invasive mesenchymal breast cancer cells caused a phenotypic switch reminiscent of a mesenchymal-to-epithelial transition (MET) characterized by changes in the cytoskeletal architecture, reexpression of hMENA(11a), and a reduction in cell invasion. hMENA-positive primary breast tumors, which are hMENA(11a)-negative, are more frequently E-cadherin low in comparison with tumors expressing hMENA(11a). These data suggest that polarized and growth-arrested cellular architecture correlates with absence of alternative hMENA isoform expression, and that the hMENA splicing program is relevant to malignant progression in invasive disease.