Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer (BC) growth, nothing is known whether ISO possesses an inhibitory effect on BC invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse invasive BC development following bladder carcinogen N-butyl-N- (4-hydroxybutyl) nitrosamine (BBN) exposure in vivo. We also found that ISO suppressed human BC cell invasion accompanied by up-regulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro. Accordingly, FOXO1 was profoundly down-regulated in human BC tissues, and was negatively correlated with BC invasion. Forced expression of FOXO1 specifically suppressed high grade human BC cell invasion, while knockdown of FOXO1 promoted non-invasive BC cells becoming invasive BC cells. Moreover, knockout of FOXO1 significantly increased BC cell invasion and abolished the ISO inhibition of invasion in human BC cells. Further studies showed that the inhibition of STAT1 phosphorylation at Tyr701 was crucial for ISO up-regulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse invasive BC formation. These findings not only provide a novel insight into the understanding of mechanism of BC's propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such BC invasion through targeting the STAT1-FOXO1-MMP2 axis.