This study evaluated nano-iron sulfide (nFeS) as an iron source on the bioavailability, biocompatibility, and tolerance in broilers. Broilers were allocated to the five diets: an iron-deficient basal diet (ID), the basal diet with 80 mg Fe/kg as FeSO4, or 20, 40, or 80 mg Fe/kg as nFeS. Results showed that nFeS treatments (40, 80 mg/kg) improved average daily gain and feed conversion rate compared to ID, showed comparable or even superior effects to FeSO4 on growth performance during 1-21 days (p < 0.05). nFeS improved whole blood Hb content and PCV percentage at day 21, while increasing serum SOD and GSH-Px activities at day 42. nFeS (40, 80 mg/kg) had or tended to deposit higher iron content in liver, spleen, duodenum and serum than FeSO4 groups. nFeS significantly increased breast meat lightness and redness compared with the ID (p < 0.05) and showed comparable effects on postmortem pH, drip loss, and shear force relative to FeSO₄. nFeS reshaped the microbial composition in the jejunum and cecum, particularly enriching the abundance of beneficial bacteria such as Lactobacillus in the jejunum. At the dose up to 400 mg/kg, nFeS revealed no negative effects in broilers. In conclusion, nFeS is an effective iron supplement with high bioavailability and comparability, and additional benefit in shaping intestinal microbiota.