Spiders belonging to the genus Phoneutria (Perty, 1833), most commonly known as ‘armed’ spiders, are among the most dangerous species in Brazil due to high toxicity of their venom, associated with their habit of invading domestic or specific areas such as banana plantations. The venom of Phoneutria spiders is secreted by a pair of venom glands located inside their cephalothoraxes and connected to the chelicerae by two independent ducts. In the present study, the microanatomy and histological structure of the venom glands of Phoneutria nigriventer (Keyserling, 1891) were examined in detail by histochemical and conventional stains with laser confocal, scanning and transmission electron microscopies. The analysis confirmed the bulbous-shaped organ previously observed by others. The venom glands of P. nigriventer are covered externally by a double layer of striated muscles, which are arranged in a spiral fashion. This disposition of the external muscle fibers might provide the contraction movement of the venom gland to release their contents during a sting aggression. The presence of pore-like openings between the muscle fibers that cover the venom glands of P. nigriventer was considered quite remarkable. The presence of axon-like structures between the muscle fibers seen in the gland surface was also quite remarkable. The secretory epithelium of P. nigriventer invaginates into the gland lumen, contributing to the increase of the secretory surface area and also accommodating a higher number of secretory cells. Our observation of histological sections and SEM showed that the secretory cells in the venom gland of P. nigriventer form complex structures, secretory units, which originate at the base near the muscular layer and that extend into the central area until the gland lumen. Our study also identified a possible holocrine secretory mechanism of P. nigriventer venom gland, at least in the first venom milking, since we were able to see nuclei stained on confocal laser microscopy. However, our observation cannot disregard other possible types of secretory pathways in subsequent milkings, since we found no nuclei in the second and in the third venom secretions.