Herein, we describe the complete stereostructure, synthesis, and biological properties of kalkitoxin (1), a novel neurotoxic lipopeptide from a Caribbean collection of Lyngbya majuscula. The toxic metabolite kalkitoxin (1) was isolated via sequential silica gel VLC, column chromatography (CC), and normal-phase HPLC guided by brine shrimp and fish toxicity assays, and re-isolated through bioassay-guided fractionation using rat neuron primary cell cultures or inhibition of IL-1β-stimulated secretory phospholipase A2 (sPLA2) in hepatocarcinoma cells. Structural elucidation of kalkitoxin (C21H38N2OS) was performed using 13C NMR, E.COSY, HSQC, and modified HSQMBC experiments, revealing four degrees of unsaturation (two double bonds, one carbonyl, one ring). The C3 stereochemistry was determined as R via Marfey's analysis, while the relative stereochemistry of C7, C8, and C10 was established using the J-based configuration approach. To confirm the absolute stereochemistry, all possible configurations of kalkitoxin were synthesized, and (3R,7R,8S,10S,2′R)-kalkitoxin was found identical to the natural product. Biological evaluations showed kalkitoxin exhibits potent brine shrimp toxicity (LC50 170 nM), goldfish ichthyotoxicity (LC50 700 nM), exceptional neurotoxicity to rat neurons (LC50 3.86 nM, reversible by NMDA receptor antagonists), inhibition of IL-1β-induced sPLA2 secretion in HepG2 cells (IC50 27 nM), and potent blockade of voltage-sensitive Na+ channels in mouse neuro-2a cells (EC50 1 nM, more potent than saxitoxin with EC50 8 nM). Synthetic (3R,7R,8S,10S,2′R)-kalkitoxin matched the natural product in potency for brine shrimp toxicity, while its enantiomer was significantly less active (LC50 9300 nM).