LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, has been widely used to study the function of PI3K in cellular responses. Based on its inhibitory effect on PI3K, LY294002 has been shown to exert antitumorigenic effect in vivo and in vitro. Here, we report that LY294002 alters early growth response 1 (EGR-1) phosphorylation and subsequently enhances activating transcription factor 3 (ATF3) expression independently of PI3K inhibition. This pathway may be, in part, responsible for the antitumorigenic effect of LY294002 in human colorectal cancer cells. ATF3 expression was increased by LY294002, followed by the induction of apoptosis in several colorectal cancer cell lines. This is consistent with results showing that the down-regulation of the ATF3 gene by small interfering RNA suppressed LY294002-induced apoptosis in HCT-116 cells. On the other hand, ATF3 expression was not affected by another PI3K inhibitor, wortmannin, as well as phosphatase and tensin homologue or dominant-negative Akt overexpression. We also found that LY294002 increases ATF3 promoter activity and the transactivation is partly mediated by a GC-rich sequence located in the promoter. EGR-1 binds to the ATF3 promoter as assessed by gel shift assay. Furthermore, phosphorylated EGR-1 was highly increased in LY294002-treated cells, indicating that EGR-1 phosphorylation induced by LY294002 may facilitate ATF3 transactivation. Our data suggest that EGR-1 acts as a mediator in LY294002-induced ATF3 expression via a PI3K-independent pathway. ATF3 and EGR-1 may provide a novel explanation for the antitumorigenic properties of LY294002 in human colorectal cancer cells.