oskar messenger RNA localization at the posterior pole of the Drosophila oocyte is essential for germline and abdomen formation in the future embryo. The nuclear shuttling proteins Y14/Tsunagi and Mago nashi are required for oskar mRNA localization, and they co-localize with oskar mRNA at the posterior pole of the oocyte. Their human homologues, Y14/RBM8 and Magoh, are core components of the exon-exon junction complex (EJC). The EJC is deposited on mRNAs in a splicing-dependent manner, 20-24 nucleotides upstream of exon-exon junctions, independently of the RNA sequence. This indicates a possible role of splicing in oskar mRNA localization, challenging the established notion that the oskar 3' untranslated region (3'UTR) is sufficient for this process. Here we show that splicing at the first exon-exon junction of oskar RNA is essential for oskar mRNA localization at the posterior pole. We revisit the issue of sufficiency of the oskar 3'UTR for posterior localization and show that the localization of unrelated transcripts bearing the oskar 3'UTR is mediated by endogenous oskar mRNA. Our results reveal an important new function for splicing: regulation of messenger ribonucleoprotein complex assembly and organization for mRNA cytoplasmic localization.