Nobox is a homeobox gene expressed in oocytes and critical in oogenesis. Nobox deficiency leads to rapid loss of postnatal oocytes. Early oocyte differentiation is poorly understood. We hypothesized that lack of Nobox perturbs global expression of genes preferentially expressed in oocytes as well as microRNAs. We compared Nobox knockout and wild type ovaries using Affymetrix 430 2.0 microarray platform. We discovered that 28 out of 38 (74%) of the genes down-regulated more than five fold in the absence of Nobox were preferentially expressed in oocytes, while only 5 out of 33 (15%) of genes up-regulated more than five fold in the absence of Nobox, were preferentially expressed in oocytes. Protein binding microarray helped identify nucleotide motifs that NOBOX binds, and that several down-regulated genes contain within putative promoter regions. MicroRNA population in newborn ovaries deficient of Nobox, was largely unaffected. Genes whose proteins are predicted to be secreted, but previously unknown to be significantly expressed in early oogenesis, were down regulated in Nobox knockouts and included astacin-like metalloendopeptidase (Astl), Jagged 1 (Jag1), oocyte secreted protein 1 (Oosp1), fetuin beta (Fetub) and R-spondin 2 (Rspo2). In addition, pluripotency associated genes, Pou5f1 and Sall4 are drastically down-regulated in Nobox deficient ovaries, while testes determining gene Dmrt1 is over-expressed. Our findings indicate that Nobox is likely an activator of oocyte-specific gene expression, and suggest that oocyte plays an important role in suppressing expression of male determining genes such as Dmrt1.