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Background Antikeratin (AK) autoantibodies, circulating antibodies against epidermal keratins, have been detected in all normal human sera. However, direct evidence on the biological significance of AK autoantibodies is still lacking. Objectives To purify AK autoantibodies from human serum and to make a preliminary study of their biological effects on human keratinocytes. Methods We first extracted keratin polypeptides from human stratum corneum and analysed their purity using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Next, a keratin affinity column was prepared with the extracted keratins, and AK autoantibodies were purified from pooled normal human serum. Antibodies obtained were identified with SDS–PAGE, enzyme-linked immunosorbent assay, immunoperoxidase staining, immunoelectron microscopy and Western blotting. The biological effect of AK autoantibodies on cultured human keratinocytes was studied using a DNA synthesis assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric determination and cell cycle analysis. Results On average, 1·83 ± 0·24 mg of antibodies could be purified from 10 mL of pooled human serum. High-titre IgG (about 1 : 70) and low-titre IgM (about 1 : 30) AK autoantibodies were obtained. The DNA synthesis assay and MTT colorimetric determination demonstrated that AK autoantibodies have a significant dose-dependent inhibitory effect on cultured keratinocytes. Correlation coefficients in the two experiments were − 0·583 and − 0·797, respectively. Cell cycle analysis indicated that a small dose of AK autoantibodies leads to inhibition of proliferation of cultured keratinocytes, whereas a large dose of AK autoantibodies causes a visible hypodiploid peak, suggesting apoptosis of keratinocytes. Conclusions The present research lays a solid foundation for further investigation into the biological significance of natural AK autoantibodies.