Initially trained by his father, Jacques de Gheyn II moved to Haarlem around 1585 to study with Hendrick Goltzius for five years. He absorbed Goltzius’s sinuous linear technique, which appeared in de Gheyn’s early engravings. He moved to Leiden in the mid-1590s, then gave up engraving around 1600 and began painting and experimenting with etching. By 1605 de Gheyn had settled in The Hague, where he regularly worked for Holland's rulers. He designed Prince Maurice's garden, which included the Netherlands's first grottoes.

Along with Goltzius, de Gheyn created some of Dutch art’s earliest female nudes. He also painted some of Holland's earliest Vanitas still lifes and flower paintings. He made over 1,500 innovative drawings, including many landscapes and natural history illustrations. Particularly through his scores of prints and drawings, de Gheyn helped lead Dutch art from its decorative Mannerist style to naturalism.