The tripartite motif (TRIM)-containing proteins are a diverse family of proteins that are involved in the regulation of innate immune responses. TRIM39 is a member of the TRIM family and contains E3 ubiquitin ligase activity. In this study, a TRIM39 homolog from the Chinese softshell turtle (Pelodiscus sinensis), PsTRIM39, was identified, and its functional characterization was investigated. PsTRIM39 is a protein of 470 amino acids containing a conserved RING-finger domain, B-BOX domain, PRY domain and SPRY domain in the TRIM family. Sequence structure and phylogenetic analysis indicated PsTRIM39 has the closest relationship with that of birds. Transcriptional profiling analysis revealed that PsTRIM39 mRNA was upregulated after challenge with Aeromonas hydrophila or the soft-shelled turtle virus, iridovirus. The subcellular localization of PsTRIM39 was in the cytoplasm, which is similar to that of fish. Furthermore, PsTRIM39 colocalized with lysosomes in Fathead minnow (FHM) cells, indicating that it may play a role in immune-related function. An NFκB functional assay showed that overexpression of PsTRIM39 enhanced NFκB activity in FHM cells, which is different from that of mammalian TRIM39. Taken together, these results provide, for the first time, the structural and functional characterization of a TRIM family member in the innate immune responses of reptiles and suggest that PsTRIM39 has distinct evolutionary properties representing the transitional stage from lower vertebrates to higher vertebrates in evolution.