The endocytic adaptor protein Numb has a major role in development as an intrinsic regulator of cell fate determination and inhibitor of the Notch signaling pathway. In vertebrates, four protein isoforms of Numb are produced through alternative splicing (AS) of two cassette exons (exons 3 and 9). AS of coding exon 9 (E9) produces E9-included (p72/p71) and-excluded (p66/p65) protein products. Expression of Numb isoforms is developmentally regulated and E9-included products are expressed in progenitors, whereas E9-excluded isoforms are dominantly expressed in differentiated cells. Analyses of AS events in multiple cancers previously identified a switch in Numb transcript and protein expression from the E9-excluded to the E9-included isoform, suggesting that misregulation of the mechanisms that control E9 inclusion may have a role in tumorigenesis. Here we identify splicing factors ASF/SF2 and PTBP1 as regulators of E9 splicing and show that activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway promotes E9 inclusion in cancer cells. Our evidence supports a mechanism by which Numb AS is regulated in response to oncogenic signaling pathways, and contributes to activation of downstream pathways to promote tumorigenesis.