We used a combination of morphological, molecular and biological data to characterize a novel Henneguya (Myxozoa) species infecting the Amazonian prochilodontid Semaprochilodus insignis or “kissing prochilodus”, a popular food fish and aquarium species in the Brazilian Amazon. Twenty-one S. insignis were caught live from the Tapajós river, Pará State, Brazil, then examined for myxozoan infections. Cysts of a novel Henneguya species were observed in the connective tissue of the fins. Myxospores measured 48 ± 4.9 (39.5–60.8) µm total length, of which caudal appendages were 33 ± 4.5 (26.4–45.2) µm and spore body was 15 ± 1.6 (12.4–20.5) µm. The spore body was 4.0 ± 0.6 (2.7–5.3) µm wide × 3.2 ± 0.4 (2.7–3.6) µm thick, with two unequal polar capsules (nematocysts) 7.2 ±0.8 (5.2–8.3) × 1.5 ± 0.3 (1.0–2.2) µm for the larger capsule and 5 ± 0.7 (4.0–6.3) × 1.4 ± 0.2 (1.0–1.8) µm for the smaller capsule. Polar tubules had 8–13 turns. Generative cells, immature and mature myxospores were observed within plasmodia. Ultrastructure showed plasmodia surrounded by collagen fibers, with the plasmodial membrane having pinocytotic channels. Phylogenetic analysis of small subunit ribosomal DNA sequences showed that the new Henneguya species clustered as a sister taxon to Henneguya tietensis, a parasite of the gills of the prochilodontid fish Prochilodus lineatus, from the geographically distant Paraná–Paraguai River basin.