Updated project metadata. Aims: to evaluate the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects of the antioxidant and disease-associated human flavoprotein NQO1. Results: in vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by HDX in different ligation states provided insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. Innovation: our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale. Conclusions: we show that site-specific phosphorylation of human NQO1 may cause pleitropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease.