To better understand the role of PRL-3 in progression and metastasis of colorectal cancer (CRC), we searched for PRL-3 associated proteins using proteomic methods. We identified 39 PRL-3 associated proteins based on proteomic strategy. Stathmin, a key oncoprotein, was proved to be a new PRL-3 associated protein. Notably, co-immunoprecipitation assays in both endogenous CRC cell lines and CRC tissues indicated that PRL-3 could interact with stathmin. And, both stathmin and PRL-3 contributed to microtubule (MT) destabilization of CRC cells. Moreover, gain-of-function and loss-of-function analyses revealed that stathmin promoted proliferation, cell adhesion, and migration of human CRC cells. Immunohistochemical analysis of 149 colorectal tumor samples showed that overexpression of stathmin was strongly correlated with tumor differentiation (P = 0.035), tumor invasion (P = 0.024), lymph node status (P < 0.001), Dukes classification (P < 0.001), and TNM staging (P < 0.001) of CRC patients. Univariate and multivariate survival analyses further supported that overexpression of stathmin protein was a potential independent poor prognostic factor for CRC. Our results reveal many PRL-3 associated proteins for the first time. The oncoprotein stathmin plays a key role in CRC as a new target of PRL-3. Interaction between PRL-3 and stathmin leads to MT destabilization of CRC cells, which contributes to progression and metastasis of CRC.