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Metastatic colonization is an ominous feature of cancer progression. Recent studies have established the importance of pre-mRNA alternative splicing (AS) in cancer biology. However, little is known about the transcriptome-wide landscape of AS associated with metastatic colonization. Both in vitro and in vivo models of metastatic colonization were utilized to study AS regulation associated with cancer metastasis. Transcriptome profiling of prostate cancer cells and derivatives crossing in vitro or in vivo barriers of metastasis revealed splicing factors with significant gene expression changes associated with metastatic colonization. These include splicing factors known to be differentially regulated in epithelial–mesenchymal transition (ESRP1, ESRP2, and RBFOX2), a cellular process critical for cancer metastasis, as well as novel findings (NOVA1 and MBNL3). Finally, RNA-seq indicated a large network of AS …