The secreted molecule osteopontin (OPN) is important for metastasis by various cancers. While the full-length form, OPN-a, is also present in healthy tissues, human breast tumors generate a shorter splice form, OPN-c, which has only been found in cancers. OPN-c is more potent than OPN-a in supporting anchorage-independence and may be critical for metastasis. Here we analyze the structural requirements for OPN-c function. An antibody specific to the splice junction neutralizes the OPN-c effects. A short peptide covering the sequence around the splice junction inhibits, whereas a long peptide mimics the effects of OPN-c. Site-directed mutagenesis identifies the distal, but not the proximal phosphorylation sites upstream and downstream of the splice junction as important for function. The interposed amino acids are also important as their mutation to alanines leads to a loss of OPN-c effects. We find that small peptides and specific antibodies can neutralize the anchorage-independence enhancing functions of OPN-c. This implies potential for future therapeutic approaches. © 2013 Wiley Periodicals, Inc.