Semaphorin 4B (SEMA4b) has been shown to play a substantial role in the invasion of non-small cell lung cancer (NSCLC). However, the regulation mechanism of SEMA4b is largely elusive. Here, we reported significant decrease in SEMA4b level and significant increase in MMP9 level in NSCLC compared with the adjacent normal tissue from the same patient. We thus used two human NSCLC lines, A549 and Calu-3, to examine whether SEMA4b may affect the expression of MMP9 in NSCLC. We found that overexpression of SEMA4b significantly decreased MMP9 level, while SEMA4b inhibition significantly increased MMP9 level. The adapted MMP9 levels affected the potential of NSCLC invasiveness correspondingly. To define the signal transduction cascades downstream of SEMA4b for regulation of MMP9 expression, we inhibited PI3K, ERK/MAPK, or JNK signaling pathway in SEMA4b knockout NSCLC and found that only inhibition of PI3K signaling pathway significantly decreased MMP9 activation. Our data thus suggest that SEMA4b may activate PI3K signaling pathway to promote MMP9 expression, which subsequently increases metastasis of NSCLC. Our study thus highlights SEMA4b as a novel therapeutic target for NSCLC.