Updated project metadata. Spiders are insect predators and their venoms are important to prey capture. Spider venomshave several potential applications as pharmaceutical compounds and insecticides. However,transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with serine peptidase inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with inhibitory activity were confirmed by mass spectrometry, identifying 6 molecules with inhibitor potential. The inhibitor Nc6834 was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor Nc6834 structurally belongs to theMIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the reactive site inhibitor residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders.