PRIMA-1(Met) is the methylated PRIMA-1 (p53 reactivation and induction of massive apoptosis) and could restore tumor suppressor function of mutant p53 and induce p53 dependent apoptosis in cancer cells harboring mutant p53. However, p53 independent activity of PRIMA-1(Met) remains elusive. Here we reported that PRIMA-1(Met) attenuated colorectal cancer cell growth irrespective of p53 status. Kinase profiling revealed that mitogen-activated or extracellular signal-related protein kinase (MEK) might be a potential target of PRIMA-1(Met). Pull-down binding and ATP competitive assay showed that PRIMA-1(Met) directly bound MEK in vitro and in cells. Furthermore, the direct binding sites of PRIMA-1(Met) were explored by using a computational docking model. Treatment of colorectal cancer cells with PRIMA-1(Met) inhibited p53-independent phosphorylation of MEK, which in turn impaired anchorage-independent cell growth in vitro. Moreover, PRIMA-1(Met) suppressed colorectal cancer growth in xenograft mouse model by inhibiting MEK1 activity. Taken together, our findings demonstrate a novel p53-independent activity of PRIMA-1(Met) to inhibit MEK and suppress colorectal cancer growth.