Freshwater habitats of the Neotropics exhibit a gradient from relatively neutral, ion-rich whitewater to acidic, ion-poor blackwater. Closely related species often show complementary distributions among ionic habitats, suggesting that adaptation to divergent osmoregulatory environments may be an important driver of Neotropical fish diversity. However, little is known about the evolutionary tradeoffs involved in ionoregulation across distinct freshwater environments. Here, we surveyed gill mRNA expression of Cichla ocellaris var. monoculus, a Neotropical cichlid, to examine cellular and physiological responses to experimental conditions mimicking whitewater and blackwater. Gene ontology enrichment of expressed genes indicated that the gills were remodeled during both forms of environmental challenge, with changes biased towards the cellular membrane. We observed expression of signaling pathways from both the acute and extended response phases, including evidence that growth hormone (GH) may mediate osmoregulation in whitewater through paracrine expression of insulin-like growth factor I (IGF-I), but not through the GH receptor, which instead showed correlated up-regulation with the prolactin receptor and insulin-like growth factor II in blackwater. Differential expression of genes related to paracellular tight junctions and transcellular ion transport showed responses similar to euryhaline fishes in fresh versus seawater, with some exceptions, suggesting that relaxed ion retention via the gills, possibly mediated by the GH/IGF-I axis, is a strong candidate for evolutionary modification in whitewater and blackwater endemic populations. In each osmoregulatory domain, we saw examples of contrasting differential expression of paralogs of genes that are single copy in most terrestrial vertebrates, indicating that adaption by fishes to diverse physicochemcial environments has capitalized on diversification of osmoregulatory gene families.