Background <p>The family of protein disulfide isomerases (PDIs) are thiol oxidoreductases located predominantly in the endoplasmic reticulum that catalyze thiol-disulfide exchange for normal protein folding. Recent studies have shown that this family enzymes such as PDI contribute to the meiosis of spermatocytes and male fertility. However, the role of PDIA6 in the PDI family in spermatogenesis has not been characterized using genetically modified animal models.</p> Methods <p>A premeiotic PDIA6 conditional knockout (<i>Stra8-Cre/Pdia6</i>^<i>fl/fl</i>) mouse model was generated for showing an essential role for PDIA6 in male fertility. To investigate the mechanism underlying the role of PDIA6 in this process, we performed a series of experiments including fertility assessment, scanning and transmission electron microscopy, TUNEL assay, spermatocyte spreading, immunofluorescence staining, intracellular calcium concentration measurement, 3-(N-maleimide-propionyl) biocytin (MPB) labeling and protein quantitative mass spectrometry.</p> Results <p>Abnormal round-headed shape resembling partial globozoospermia was observed in <i>Stra8-Cre/Pdia6</i>^<i>fl/fl</i> mice. Ultrastructural analysis of PDIA6-deficient sperm demonstrated acrosome fragmentation and detachment from the nucleus, disrupted acroplaxome structure, disorganized flagellar axonemes, and cytoplasmic retention. PDIA6 deficiency also impaired the sperm acrosome reaction and calcium mobilization. Proteomic profiling revealed downregulation of acrosomal membrane and vesicle proteins, as well as calcium channel complexes in PDIA6-deficient testes. Moreover, PDIA6 deficiency down-regulated the synthesis of zona pellucida binding protein (ZPBP) in testes by impairing disulfide bond formation, and induced endoplasmic reticulum stress and apoptosis.</p> Conclusions <p>PDIA6 is essential for redox regulation of protein synthesis and spermatogenesis causing male fertility in mice.</p>