Spermatid‐specific thioredoxin‐1 (Sptrx‐1) is the first member of the thioredoxin family of proteins with a tissue‐specific expression pattern, found exclusively in the tail of elongating spermatids and spermatozoa. We describe here further biochemical characterization of human Sptrx‐1 protein structure and enzymatic activity. In gel filtration chromatography human Sptrx‐1 eluates as a 400 kDa protein consistent with either an oligomeric form, not maintained by intermolecular disulfide bonding, and/or a highly asymmetrical structure. Analysis of circular dichroism spectra of fragments 1–360 and 361–469 and comparison to spectra of full‐length Sptrx‐1 supports a two‐domain organization with a largely unstructured N‐terminal domain and a folded thioredoxin‐like C‐terminal domain. Functionally, Sptrx‐1 behaves as an oxidant in vitro when using selenite, but not oxidized glutathione, as electron acceptor. This oxidizing enzymatic activity suggests that Sptrx‐1 might govern the stabilization (by disulfide cross‐linking) of the different structures in the developing tail of spermatids and spermatozoa.