UNLABELLED: Sea anemones represent one of the emerging groups of interest concerning venomous animals in toxinology and the goal of the present work was the prospection, and the structural and functional characterization of the compounds present in the secretion of the sea anemone Stichodactyla duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low molecular weight fractions were further submitted to RP-HPLC resulting in 35 new subfractions that were subsequently analyzed by offline MALDI-TOF mass spectrometry. MALDI peptide mass fingerprinting yielded up to 134 different molecular masses, ranging from m/z 901 to 10,833. Among these subfractions, a new peptide of 3431Da, named U-SHTX-Sdd1, was purified and completely sequenced by automated Edman's degradation and tandem mass spectrometry. An analysis of U-SHTX-Sdd1 revealed a modified O-HexNAc-Threonine at position 1, which, at the best of our knowledge, constitutes the first sea anemone toxin reported with such post-translational modification. Because of its sequence similarity with other sea anemone toxins, the pharmacological activity of U-SHTX-Sdd1 was assessed by electrophysiological measurements using the two electrode voltage-clamp technique on cloned voltage-gated potassium channel subtypes, expressed in Xenopus laevis oocytes. However, U-SHTX-Sdd1 did not show activity on these channels. A large-scale proteomic approach was also employed to shed lights on the sea anemone compounds, and a total 67 proteins and peptides were identified. BIOLOGICAL SIGNIFICANCE: In this manuscript, we report an extensive characterization of S. duerdeni secretion by means of peptide mass fingerprinting and high-throughput proteome analyses. Also, we report the structure of a new glycopeptide by a combination of biochemical techniques. Despite the previous studies that described proteinaceous compounds present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy of analysis is perfectly suitable to other sea anemone secretions and animal venoms. Moreover, new peptide structures can arise contributing to the knowledge of the diversity of these animal peptides.