We recently identified an additional isoform of human thymosin beta 15 (also known as NB-thymosin beta, gene nameTMSB15A) transcribed from an independent gene, and designatedTMSB15B. The purpose of this study was to investigate whether these isoforms were differentially expressed and functional. Our data show that theTMSB15AandTMSB15Bisoforms have distinct expression patterns in different tumor cell lines and tissues.TMSB15Awas expressed at higher levels in HCT116, DU145, LNCaP, and LNCaP-LN3 cancer cells. In MCF-7, SKOV-3, HT1080, and PC-3MLN4 cells,TMSB15AandTMSB15Bshowed approximately equivalent levels of expression, whileTMSB15Bwas the predominant isoform expressed in PC-3, MDA-MB-231, NCI-H322, and Caco-2 cancer cells. In normal human prostate and prostate cancer tissues,TMSB15Awas the predominant isoform expressed. In contrast, normal colon and colon cancer tissue expressed predominantlyTMSB15B. The two gene isoforms are also subject to different transcriptional regulation. Treatment of MCF-7 breast cancer cells with transforming growth factor beta 1 repressedTMSB15Aexpression but had no effect onTMSB15B. siRNA specific to theTMSB15Bisoform suppressed cell migration of prostate cancer cells to epidermal growth factor, suggesting a functional role for this second isoform. In summary, our data reveal different expression patterns and regulation of a new thymosin beta 15 gene paralog. This may have important consequences in both tumor and neuronal cell motility.