ERp57/PDIA3/1,25-MARRS has diverse functions and multiple cellular locations in various cell types. While classically described as an endoplasmic reticulum (ER) resident protein, ERp57 has a nuclear location sequence (NLS) and can enter the nucleus from the cytosol to alter transcription of target genes. Dysregulation and variable expression of ERp57 is associated with a variety of cancers including hepatocellular carcinoma (HCC). We investigated the dynamic mobility of ERp57 in an HCC cell line, HepG2, to better understand the movement and function of the non-ER resident pool of ERp57. Subcellular fractionation indicated ERp57 is highly expressed in the ER with a smaller cytoplasmic pool in HepG2 cells. Utilizing an ERp57 green fluorescent protein fusion construct created with and without a secretory signal sequence, we found that cytoplasmic ERp57 translocated to the nucleus within 15 min after tumor necrosis factor-α (TNF-α) treatment. Protein kinase C activators including 1,25-dihydroxyvitamin D3 and phorbol myristate acetate did not trigger nuclear translocation of ERp57, indicating translocation is PKC independent. To determine if an interaction between the rel homology binding domain in ERp57 and the nuclear factor-κB subunit, p65, occurred after TNF-α treatment and could account for nuclear movement, co-immunoprecipitation was performed under control and conditions that stabilized labile disulfide bonds. No support for a functional interaction between p65 and ERp57 after TNF-α treatment was found in either case. Immunostaining for both ERp57-GFP and p65 after TNF-α treatment indicated that nuclear translocation of these two proteins occurs independently in HepG2 cells. J. Cell. Biochem. 112: 2606–2615, 2011. © 2011 Wiley-Liss, Inc.