Abstract: The aim of this study was to evaluate the expression level of MARCKS in prostate cancer cell lines, construct expression plasmids of MARCKS, explore the expression of plasmids in cells and the subcellular localization of the exogenous proteins, and detect the effects of overexpression of MARCKS gene on the migration ability of PC-3 cells. Western blot was carried out to evaluate the level of MARCKS in several prostate cancer cell lines. N or C terminal HA tagged primers were designed according to the coding sequence of MARCKS. The target fragment was amplified by PCR and subjected to ligate with vector. After enzyme digestion identification and sequence alignment, the plasmids with correct sequence were transfected into PC-3 cells. Western blot was used to detect N-HA-MARCKS and C-HA-MARCKS proteins, and immunofluorescence staining was utilized to detect their subcellular localizations. Transwell assay was used to evaluate the effects of N-HA-MARCKS and C-HA-MARCKS on the migration ability of PC-3 cells. In this study, the expression plasmids of MARCKS gene with different tag were constructed. The molecular mass of C-HA-MARCKS protein was larger than N-HA-MARCKS, and C-HA-MARCKS protein was more obviously located on the cell membrane. Both N-HA-MARCKS and C-HA-MARCKS promoted the migration ability of PC-3 cells. Our study provides an experimental basis for further elucidating the molecular mechanism of MARCKS in prostate cancer cells.