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Recent studies showed the potential of stable isotopes of the macronutrient calcium (δ⁴⁴/⁴⁰Ca) and nonessential strontium (δ⁸⁸/⁸⁶Sr) as new trophic level indicators in terrestrial vertebrates and marine teleost fishes. In this study, we tested whether similar Ca and Sr isotopic fractionation trends existed in macroinvertebrate-dominated stream food webs compared to vertebrates despite their physiological differences. We have determined the δ⁴⁴/⁴⁰Ca and δ⁸⁸/⁸⁶Sr values as well as the ⁸⁷Sr/⁸⁶Sr ratios of stream macroinvertebrates and small gobies and their potential metal sources (stream water, periphyton, and terrestrial plant litter) in upper and lower reaches of two streams in the Lake Biwa catchment, central Japan. The ⁸⁷Sr/⁸⁶Sr ratios revealed that stonefly nymphs, crustacea, and gobies mostly relied on aquatic Sr sources. Higher ⁸⁷Sr/⁸⁶Sr ratios of some crane fly and caddisfly larvae, mayfly, dobsonfly, and dragonfly nymphs indicated greater terrestrial contributions via plant litter. Positive correlations between the δ⁴⁴/⁴⁰Ca and δ⁸⁸/⁸⁶Sr values implied that similar Ca and Sr sources existed, and that Ca and Sr stable isotopes underwent similar fractionation trends although Sr was not essential. The δ⁴⁴/⁴⁰Ca and partly the δ⁸⁸/⁸⁶Sr values were positively correlated with Sr/Ca ratios and negatively with δ¹⁵N values indicating trophic effects on Ca and Sr stable isotopes. The enrichment of ⁴⁴Ca and ⁸⁸Sr in large filter-feeding caddisfly larvae was a notable exception from these trophic trends. Our data confirm that the trophic ⁴⁴Ca and ⁸⁸Sr depletion observed for marine teleost fishes and terrestrial vertebrates also applied to macroinvertebrate-dominated stream food webs despite their different physiologies indicating that shared mechanisms of Ca and Sr isotopic fractionation may exist at the cellular or molecular level between these taxa.