Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in the nitrosative stress responses in Saccharomyces cerevisiae. It is still unclear how S. cerevisiae can withstand this NO level in the absence of flavohemoglobin. To better understand the physiological function of flavohemoglobin in yeast, in the present study a label-free differential proteomics study has been carried out in wild-type and YHB1 deleted strains of S. cerevisiae grown under fermentative conditions. From the analysis, 417 proteins in Y190 and 392 proteins in ΔYHB1 were identified with high confidence. Interestingly, among the differentially expressed identified proteins, 40 proteins were found to be downregulated whereas 41 were found to be upregulated in ΔYHB1 strain of S. cerevisiae (p value < 0.05). The differentially expressed proteins were also classified according to gene ontology (GO) terms. The most enriched and significant GO terms included nitrogen compound biosynthesis, amino acid biosynthesis, translational regulation, and protein folding. Interactions of differentially expressed proteins were generated using Search Tool for the Retrieval of Interacting Genes (STRING) database. This is the first report which offers a more complete view of the proteome changes in S. cerevisiae in the absence of flavohemoglobin.