U1 small nuclear RNA (U1 snRNA), as one of the most abundant ncRNAs in human cells, plays an important role in splicing of pre-mRNAs. Compared to previous studies which have focused on the primary function of U1 snRNA and the neurodegenerative diseases caused by abnormalities of U1 snRNA, this study is to investigate how U1 snRNA over-expression affects the expression of mammal genes on a genome-wide scale. By comparing the gene expression profiles of U1 snRNA over-expressed cells with those of their controls using microarray experiments, 916 genes or loci were identified significantly Differentially Expressed (DE). These 595 up-regulated DE genes and 321 down-regulated DE genes were analyzed using annotations from GO categories and pathways from the KEGG database. As a result, three of 12 enriched pathways were well-known cancer pathways, while the other nine pathways were associated to cancers in previous studies. The further analysis of 73 genes involved in 12 pathways suggested that U1 snRNA could regulate cancer gene expression. The microarray data under the GEO Series accession number GSE84304 is available in the NCBI GEO database.