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Background Ferritin plays a key role in the development of prostate cancer (PCa). Our earlier studies showed that the knockdown of ferritin heavy chain (FTH) suppressed the migration and invasion of the prostate cancer cell line (PC3). However, the mechanisms behind FTH in the cell migration regulation of PCa have not been thoroughly investigated. Methods Isobaric tags for relative and absolute quantitation (iTRAQ) proteomics was used to analyze the protein expression in PC3 cells with FTH knockdown by small interfering RNAs and negative control cells. We subsequently ranked the differentially expressed proteins according to the change in expression. We further performed Gene Ontology (GO) analysis for the changing-expression protein. Finally, Western blot analysis was performed to determine the expression of the target protein. Results Compared with the negative group, 420 proteins were downregulated, including proteins S100A4, S100P, and S100A2, while the expression of 442 protein was elevated in FTH-silencing PC3 cells (P1.2). The mass spectrometry results showing decreased expression of protein S100A4, S100P, and S100A2 in the cells were further validated by Western blot (P1.6). Conclusions The downregulation of FTH expression reduced the level of protein S100A4, S100A2, and S100P, which all play a key role in the migration and invasion of tumor cells. Therefore, it is reasonable to assume that there are correlations between the expression of the S100A4, S100A2, and S100P genes with FTH. Based on this research, FTH may be a new biomarker for the diagnosis of PCa.