Human T lymphotropic virus type 1 (HTLV-1) requires regulated gene expression from unspliced and alternatively spliced transcripts for efficient replication and persistence. HTLV-1 Rex is known to facilitate cytoplasmic export of unspliced, gag/pol and incompletely spliced env mRNAs, but its contribution to the expression of other viral transcripts has not been experimentally assessed. In this study, we utilized HTLV-1 proviral clones, cellular fractionation, and real-time reverse transcriptase PCR to determine the role of Rex on the expression and export of all viral mRNAs. Our results indicate that the steady-state levels of the different viral mRNAs are modulated by Rex, which we attribute to a redistribution of completely spliced mRNAs toward incompletely spliced mRNAs. Furthermore, we confirmed the positive effect of Rex on the unspliced gag/pol mRNA and singly spliced env mRNA, resulting in increased cytoplasmic expression. However, the cytoplasmic export of the alternatively spliced HTLV-1 mRNAs encoding the accessory proteins and the antisense Hbz mRNA are independent of direct Rex regulation. This is consistent with the conclusion that viral mRNAs that contain the cis-acting repressive sequence (CRS) and/or a fully functional splice donor site require a Rex/RxRE interaction for efficient cytoplasmic expression.