Calcareous fens are one of the most species-rich habitats of the temperate zone of the Northern Hemisphere. In spite of this species richness, however, calcareous fens are still rather poorly investigated. Consequently, the data of the fen-associated spider fauna are also largely lacking. The aim of the research was to study the spider fauna of the calcareous fens of Latvia and to draw conclusions about what kind of spider species and ecological groups typically inhabit calcareous fen habitats. Spiders were sampled in the summer months of 2010, 2011, and 2012 at nine different calcareous fens of the coastal lowland of Latvia. The spider collection was performed by pitfall traps and a sweep net. The examined material comprised 6631 adult spider individuals representing 21 families and 149 species. The main spider ecological groups that dominated in the studied calcareous fens were hygrophilous and photophilous species which largely reflect the main properties of our studied habitats, all of which were wet, open mire habitats. Nevertheless, the fen arachnofauna consisted also of spider groups which are less typical for moist, sun-exposed, and alkaline environments, like xerophilous, sciophilous, and sphagnophilous species, respectively. Finally, several spider species collected in this study have not been previously reported for the spider fauna of Latvia, and many more might still be undiscovered in these unique and poorly investigated habitats. Therefore, it is suggested that calcareous fens deserve special attention and they should definitely be investigated further.