The concept of nanopublications was introduced by Barend Mons and Jan Velterop in 2009, in their paper Nano-Publication in the e-science era. The paper proposed atomic, machine-readable assertions with their own provenance and metadata as a new unit of scholarly communication.

Concrete nanopublications followed in subsequent years, most of them unsigned, accumulating in the first-generation nanopub-server network. The earliest dated trusty-URI nanopublications discoverable there are from 16 June 2014, part of a corpus of unsigned biomedical assertions extracted from NCBI's GeneRIF database by the Krauthammer Lab at Yale — for example, RAdBeVv56ZiRgSdbHMEk0J4C3iV5mDNBSlwo87B5P-u9c. The earliest signed entry preserved in the current (second-generation) Nanopub Registry dates from 18 August 2015: RATNbqwXNo6aO3T7BdCquQSdqZjXeisoSSTudO3qJ9KwY, an index of three example nanopubs produced while validating the signing infrastructure.