Henneguya cuniculator sp. nov. was found infecting spotted sorubim catfish Pseudoplatystoma corruscans from the São Francisco River, Minas Gerais, Brazil. The parasites form elongated plasmodia of up to 1 cm in length in the gill filaments. Mature spores were ellipsoidal from the frontal view, with total length of 29.4 ± 2.4 (mean ± SD, range 23.3-32.4) µm, body length of 12.1 ± 1.0 (10.0-14.7) µm, width of 4.8 ± 0.4 (4.0-5.9) µm, and tail length of 16.7 ± 2.0 (12.3-19.4) µm. From the lateral view, spores were biconvex, with thickness of 4.2 ± 0.7 (3.9-4.9) µm. The polar capsules were elongated and equal in size, 6.2 ± 0.3 (5.2-6.2) µm in length, and 1.8 ± 0.1 (1.4-1.9) µm in width. Ultrastructural analysis showed that the plasmodial wall had delicate projections towards the host tissue and a thin layer that prevented contact between the host cells and the parasite. In the ectoplasm, few mitochondria were observed, while generative cells, early stages of sporogenesis, and advanced spore development occurred in the plasmodial periphery, and more mature spores in internal regions. Histopathological analysis showed that plasmodia developed in the sub-epithelial connective tissue of gill filaments, causing compression of the adjacent tissues, deformation of gill filaments, and lamellar fusion. Phylogenetic analysis, based on 18S rDNA genes and using only Henneguya/Myxobolus species parasites of siluriform fish, showed grouping according to the fish family.