Intensive agriculture has degraded streams in subtropical Taiwan, but agricultural impacts on fish assemblages are not well studied. The goal of this study was to understand changes in structure and function of fish assemblages along environmental gradients in an agricultural region of South-central Taiwan. Nineteen sites in the hill and upper plain regions were selected for fish sampling during the base flow period. Water chemistry analyses and rapid habitat assessment were also conducted. Cluster analysis separated fish assemblages into four assemblage groups and a single site. A redundancy analysis (RDA) showed that environmental variables explained 73.9% of species variance. RDA axis 1 represented a habitat-diversity, cover, and nutrient gradient, whereas RDA axis 2 represented a complex riparian condition gradient. Relative abundances of dominant fish species and assemblage groups were related to water and habitat variables. Trophic and tolerance guilds were correlated with RDA axes. Number of fish species increased with decreasing elevation. Both structure and function of fish assemblages changed with water and habitat gradients in these subtropical agricultural streams.