The accumulation of microplastics (MPs) in fish gills has been widely recognized, however, whether such stress could thereby impact the physiological responses of fish gills is still unknown. Here, we investigated the impacts of three sizes (400 nm, 4 μm, 20 μm) of polystyrene (PS) MPs on (Na+, K+, Cl-) ions regulation and ammonia excretion in medaka Oryzias melastigma. Significantly increased net Na+ and K+ flux rates were observed transiently during 0-3 h and 3-9 h, but not during 9-24 h. Such results suggest that the physiological resilience of fish gills regarding Na+ and K+ regulation was unaffected upon the exposure to PS-MPs, probably evidenced by the increased secretion of mucus. However, Cl- regulation and ammonia excretion were significantly impaired, partly in consistent with the damages of ionocytes. The adverse impacts of PS-MPs on Cl- regulation and ammonia excretion were size-dependent, with significant disturbances observed in 4 μm and 20 μm treated group for Cl- regulation, but only in 20 μm treated group for ammonia excretion. The specific enrichment of Shinella and lower abundance of function profiles related to ion transport and metabolism might be responsible for the specific disturbance of Cl- regulation found in the 4 μm treated group. The enrichment of Gemmobacter also accounted for the disturbances of ammonia excretion in 20 μm treated group. Our results highlighted the impacts of PS-MPs on the physiological functions in fish gills.