Staufen1 (STAU1) is an RNA-binding protein (RBP) that interacts with double-stranded RNA structures and has been implicated in regulating different aspects of mRNA metabolism. Previous studies have indicated that STAU1 interacts extensively with RNA structures in coding regions (CDSs) and 3′-untranslated regions (3′UTRs). In particular, duplex structures formed within 3′UTRs by inverted-repeat Alu elements (IRAlus) interact with STAU1 through its double-stranded RNA-binding domains (dsRBDs). Using 3′ region extraction and deep sequencing coupled to ribonucleoprotein immunoprecipitation (3′READS + RIP), together with reanalyzing previous STAU1 binding and RNA structure data, we delineate STAU1 interactions transcriptome-wide, including binding differences between alternative polyadenylation (APA) isoforms. Consistent with previous reports, RNA structures are dominant features for STAU1 binding to CDSs and 3′UTRs. Overall, relative to short 3′UTR counterparts, longer 3′UTR isoforms of genes have stronger STAU1 binding, most likely due to a higher frequency of RNA structures, including specific IRAlus sequences. Nevertheless, a sizable fraction of genes express transcripts showing the opposite trend, attributable to AU-rich sequences in their alternative 3′UTRs that may recruit antagonistic RBPs and/or destabilize RNA structures. Using STAU1-knockout cells, we show that strong STAU1 binding to mRNA 3′UTRs generally enhances polysome association. However, IRAlus generally have little impact on STAU1-mediated polysome association despite having strong interactions with the protein. Taken together, our work reveals complex interactions of STAU1 with its cognate RNA substrates. Our data also shed light on distinct post-transcriptional fates for the widespread APA isoforms in mammalian cells.