Updated project metadata. Hyaluronidases are important venom components by acting as spreading factor of neurotoxins. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, which are the main prey organisms of spiders, are lacking. In this study, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was solved by cDNA analysis of the venom gland transcriptome and confirmed by protein analytics. Two complex N-linked glycans, which akin honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry.