Hydrogen peroxide (H2O2) is one of the key signaling factors regulating skeletal muscle adaptation to muscle contractions. Eccentric (ECC) and concentric (CONC) contractions drive different muscle adaptations with ECC resulting in greater changes. The present investigation tested the hypothesis that ECC produces higher cytosolic and mitochondrial H2O2 concentrations [H2O2] and alters gene expression more than CONC. Cytosolic and mitochondrial H2O2-sensitive fluorescent proteins, HyPer7 and MLS-HyPer7, were expressed in the anterior tibialis muscle of C57BL6J male mice. Before and for 60 min after either CONC or ECC (100 Hz, 50 contractions), [H2O2]cyto and [H2O2]mito were measured by in vivo fluorescence microscopy. RNA sequencing was performed in control (noncontracted), CONC, and ECC muscles to identify genes impacted by the contractions. [H2O2]cyto immediately after ECC was greater than after CONC (CONC: +6%, ECC: +11% vs. rest, P < 0.05) and remained higher for at least 60 min into recovery. In contrast, the elevation of [H2O2]mito was independent of the contraction modes (time; P < 0.0042, contraction mode; P = 0.4965). The impact of ECC on [H2O2]cyto was abolished by NADPH oxidase 2 (Nox2) inhibition (GSK2795039). Differentially expressed genes were not present after CONC or ECC + GSK but were found after ECC and were enriched for vascular development and apoptosis-related genes, among others. In conclusion, in mouse anterior tibialis, ECC, but not CONC, evokes a pronounced cytosolic H2O2 response, caused by Nox2, that is mechanistically linked to gene expression modifications.NEW & NOTEWORTHY This in vivo model successfully characterized the effects of eccentric (ECC) and concentric (CONC) contractions on cytosolic and mitochondrial [H2O2] in mouse skeletal muscle. Compared with CONC, ECC induced higher and more sustained [H2O2]cyto-an effect that was abolished by Nox2 inhibition. ECC-induced [H2O2]cyto elevations were requisite for altered gene expression.