SummaryOCT3/4, NANOG, SOX2 and, most recently, LIN28 have been identified as key regulators of pluripotency in mammalian embryonic and induced stem cells, and are proven to be crucial for generation of the mouse germ‐cell lineage. These factors are a hallmark of certain histological types of germ‐cell tumours (GCTs). Here, we report novel information on the temporal and spatial expression pattern of LIN28 during normal human male germ‐cell development as well as various types of GCTs. To investigate LIN28 expression, immunohistochemical analyses and quantitative proximity ligation assay‐based TaqMan protein assays were applied on snap‐frozen and formalin‐fixed, paraffin‐embedded samples as well as representative cell lines. LIN28 was found in primordial germ cells, gonocytes and pre‐spermatogonia, in contrast to OCT3/4 and NANOG, which were found only in the first two stages. LIN28 was also found in all precursor lesions (carcinoma in situ and gonadoblastoma) of type II GCTs, as well as the invasive components seminoma and the non‐seminomatous elements embryonal carcinoma and yolk sac tumour. Choriocarcinoma showed a heterogeneous pattern, while teratomas and spermatocytic seminomas (type III GCTs) were negative. This expression pattern suggests that LIN28 is associated with malignant behaviour of type II GCTs. Cell line experiments involving siRNA knockdown of LIN28, OCT3/4 and SOX2 showed that LIN28 plays a role in the maintenance of the undifferentiated state of both seminoma and embryonal carcinoma, closely linked to, and likely upstream of OCT3/4 and NANOG. In conclusion, LIN28 regulates the differentiation status of seminoma and embryonal carcinoma and is likely to play a related role in normal human germ‐cell development.