Significance Multiple studies highlight the role of various proteins in regulation of alternative splicing; however, the regulatory role of distinct posttranslational modifications during alternative splicing that contribute to tumorigenesis is enigmatic. Here we report a previously unidentified noncanonical mechanism of regulation of alternative splicing modulated by deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa) via Scaffold/matrix-associated region-binding protein 1 (SMAR1)–histone deacetylase 6 (HDAC6) complex. SMAR1 in complex with HDAC6 maintains Sam68 in a deacetylated state. We observed that ERK-1/2–dependent phosphorylation of SMAR1, knockdown of SMAR1, or loss of heterozygosity facilitates CD44 variant exon inclusion via Sam68 acetylation and thus confers invasive and metastatic potential in breast tumor cells. Our findings provide key insights into regulation of alternative splicing and the potential for therapeutic intervention during tumor metastasis.