The phenomenon of intron-mediated enhancement (IME) was discovered in 1990 based on the observation that plant introns can stimulate gene expression, particularly in monocots. However, the intrinsic mechanism of IME remains unclear because many studies have yielded various results depending on the promoter, reporter gene, flanking sequences of the intron, and target cell or tissue. In this study, the effect of the first intron of the maize ubiquitin gene (ubi1intron) was investigated by changing insertion sites, deleting specific regions and mutating individual motifs in maize (Zea maysL.) and rice (Oryza sativaL.) using ubi1intron-containing GUS(β-glucuronidase) constructs. In maize callus, the integration of the full-length ubi1intron into the GUScoding sequence at the +13, +115 and +513 positions by particle bombardment increased GUS activity approximately five-, four- and two-fold, respectively. Eight truncated ubi1introns in the pSG(13i) N construct significantly influenced GUSgene expression to different degrees in transient assays. Notably, the 3′ region deletions significantly reduced the IME effect, whereas a 142-nt deletion, pSG(13i-P5)N, in the 5′ region caused a 1.5-fold enhancement relative to pSG(13i)N. Furthermore, four site-directed mutageneses were performed in pSG(13i-P5)N; these constructs resulted in the up-regulation of GUSgene expression to different levels. The most effective modified ubi1intron, pSG(13i-M4)N, was further evaluated and proved in rice using transient experiments. In addition, the sequences flanking the GUSinsertion significantly influenced the IME effect of the vectors that were constructed. The modified ubi1intron had the potential application on crop genetic engineering.