The Itasenpara bitterling Acheilognathus longipinnis is a small cyprinid fish belonging to the Acheilognathinae, a subfamily that has an unusual symbiotic spawning relationship with freshwater mussels. During the spawning period, mature males select a mussel to accommodate spawning of a female. Because females possess short ovipositors for inserting their eggs inside the suprabranchial cavity of the host mussel via the exhalant siphon, the pattern of mussel utilization by spawning Itasenpara bitterling was investigated in the Moo River and a conservation pond (Himi City, Toyama Prefecture, Japan), so as to clarify the most preferred mussel size. In the Moo River (lotic environment), Nodularia douglasiae nipponensis (38.8% of total 747 individuals) was the major host of A. longipinnis larvae, there being no obvious size preference, possibly because neither eggs nor larvae of A. longipinnis could be readily ejected from the host in the lotic environment. By contrast, in the conservation pond (lentic environment), where N. d. nipponensis (24.3% of total 136 individuals) was again the main host of A. longipinnis larvae, a tendency to prefer smaller-sized mussels was apparent, due to the ease of ejection of eggs and larvae of A. longipinnis from larger-sized hosts (shell length >66 mm) in the lentic environment. Accordingly, differences in host mussel suitability are suggested as being related to habitat differences.