Boninite has been widely accepted to be the melting product of highly refractory mantle with the addition of slab-derived fluids. Previous studies have found that boninite, like other subduction-related magmas (e.g., mature arc basalts), has generally consistent chondrite-like iron isotope composition (c. 0.03‰), which is on average lighter than those of oceanic basalts. However, it remains unclear whether the lighter iron isotope composition of subduction-related magmas is inherited from the depleted mantle source or caused by the addition of slab-derived oxidized fluids. Moreover, boninite has highly varied element patterns (e.g., Yb, Zr), reflecting varying degrees of mantle depletion and different contributions of slab-derived fluids. Given the limited data for iron isotope composition of boninite so far available, it is thus required to systematically study iron isotope composition of boninite with different compositions to further understand the petrogenesis of boninite and the iron isotope variation of subduction-related magmas. Here we report iron isotope compositions of boninite from two classic suites on Earth, i.e., those from Bonin islands and submarine forearc (low-Ca boninite; CaO/Al2O3 mainly <0.75) and Troodos ophiolite complex, Cyprus (high-Ca boninite: CaO/Al2O3 = c. 0.85). The δ56Fe values of the Bonin boninite vary from −0.046 ± 0.003‰ to 0.078 ± 0.031‰ (2SD, SD = standard deviation of 4 times repeated analysis; with an average of 0.03 ± 0.03‰, n = 14). The δ56Fe value of the Troodos boninite is relatively constant, i.e., from 0.046 ± 0.010‰ to 0.091 ± 0.016‰ with an average of 0.06 ± 0.01‰ (n = 7). Bonin forearc basalts (FABs) were also analyzed for comparison with δ56Fe of ~0.10‰, except for those FABs with higher Th/U ratio and Cu contents (δ56Fe = 0.01–0.05‰). Together with previously reported δ56Fe data for boninite from New Caledonia (low-Ca boninite with CaO/Al2O3 = ~ 0.4, δ56Fe = ~ +0.03‰), the lower δ56Fe values of the Bonin boninite than Bonin FAB and Troodos boninite reflect the lighter iron isotope composition of the more depleted mantle source. Based on relationships between different indicators for various fluids (e.g., Ba/La, Zr/Sm) and modeling, Bonin boninite is likely produced by partial melting of the refractory mantle with an addition of ~5–10% melts from sediments and oceanic crust, while the formation of Troodos boninite can be ascribed to the melting of less refractory mantle, which has been previously metasomatized by ~5% fluids derived from oceanic crust and sediments.