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.