Abstract: Objective To construct pcDNA3.1-Rap2b eukaryotic expression vector and to understand the effects on AKT, ERK, JNK and P38 signaling pathway of NIH3T3 cells through transfecting Rap2b gene into NIH3T3 cells. Methods Eukaryotic expression vector pcDNA3.1-Rap2b was constructed and was stable transfected into NIH3T3 cell, and its effects on the AKT、ERK、JNK and P38 pathway of NIH3T3 cells were observed by Western blotting to measure the level of the expression of AKT、ERK、JNK and P38 Phosphorylation protein and total protein. Results The level of the expression of P38 Phosphorylation protein was higher in the pcDNA3.1-Rap2b vector than that of control（P＜0.05）, and the level of the expression of AKT、ERK、JNK Phosphorylation protein was not different comparing with the control（P＞0.05）. Conclusion Rap2b might activate P38 signaling pathway, but not activate AKT, ERK, JNK signaling pathway.