Flavohemoglobins and flavodiiron proteins are two families of enzymes involved in nitrosative detoxification. However, the physiological oxygen-related conditions under which they work and their relative role are still a matter of debate. To address this question we analyzed the function of the putative flavohemoprotein of Staphylococcus aureus, an organism that lacks a flavodiiron-like gene. In this report we show that the recombinant protein contains all features typical of canonical flavohemoglobins and that the transcription of flavohemoglobin gene was upregulated by nitrosative stress in an oxygen-dependent manner. However, and in contrast to other bacterial flavohemoglobins, the S. aureus protein has no apparent role in aerobic nitrosative protection, being only beneficial when cells of S. aureus are submitted to nitrosative stress in a microaerophilic environment. The in vivo data corroborates the proposal that Hmp acts physiologically as a denitrosylase.