To systematically understand the effects of the culturing properties and biodegradation reactions on 4-nitrophenol (PNP)-degrading microorganisms, the bacterium YUP-3 was isolated from soil samples with no PNP-pollution history using a novel method. This bacterium was domesticated to utilize PNP as the sole source of carbon and nitrogen in mineral salt medium. Response surface methodology (RSM) was used to select the most important variables from the simultaneous study on PNP degradation after 48 h incubation. The theoretical maximum PNP degradation of 100.0% was obtained under optimized conditions, and temperature and PNP initial concentrations were concluded to be significant variables. Under carbon- and nitrogen-deficient conditions, five metabolic products of PNP were identified through gas chromatography–mass spectrometry. Hydrogenation and deoxidation were analyzed as two main reactions through PNP degradation to produce hydroquinone. These results demonstrated the importance of the novel PNP degradation pathway under nutrition deficient conditions and the potential use of this highly effective PNP degrading bacterium.