Neuroglobin (NGB) is an oxygen-binding protein that takes part in several processes that safeguard redox homeostasis, gaining an important protective role in neuronal and extra-neuronal cells. NGB remarkably exerts its function upon up-regulation by NGB-inducible molecules such as 17β-estradiol (E2) and H2O2. Despite this, the essential mechanisms related to NGB function remain still undefined. Human MCF-7 breast cancer cells knocked out (KO) for NGB gene obtained using CRISPR/Cas9 technology were analyzed by shotgun label-free quantitative proteomics in comparison with control cells. Differential proteomics experiments were also performed using NGB-KO cells after treatment with E2, H2O2, and E2+H2O2 versus their corresponding non-treated controls.