Abstract: Introns increasing mRNA transcription have been found extensively in eukaryotes. However, not all of the introns could affect gene expression and functional profiles, especially stressrelated profiles and introns having such effect are still unclear. In this study, four intron vectors were constructed and tested in tobacco by histochemical analysis and fluorometric analysis. Results showed that the 5′ UTR intron of gene encoding the stress responsive protein AmCBL1 functionally elevated gene expression, and the orientation of 5′ UTR intron was a strong predictor of its ability to increase expression. We found that the intron could enhance transcription when inserted in both the forward and reverse orientations under several stresses and hormones. However, transcriptional enhancement of intron in the opposite orientation under normal condition was not significant. This directionality of the inserted 5′ UTR intron provides further insight into the regulation of gene expression.