Doxorubicin is an effective anticancer drug but its use is limited due to its adverse side effects such as infertility and cardiomyopathy. Some possible mechanisms of the action of doxorubicin have been postulated, but the initial gene deregulation response has not been investigated. Fetal life stages are critical periods in mammalian oogenesis. This study analyzes gene expression alterations in mouse fetal oocytes exposed in vitro to this anticancer agent. cDNA libraries were generated from isolated fetal oocytes and differential screenings performed with cDNAs from in vitro doxorubicin-treated and -untreated oocytes. Differentially expressed genes were assessed by real-time RT-PCR to quantify the extent of their transcriptional control in doxorubicin-exposed oocytes. The results show that doxorubicin alters the expression of genes involved in the mitochondrial respiratory chain, intracellular transport and cell differentiation. Finally, the up-regulation of a differentially expressed gene (metaxin) mediated by its promoter was evaluated in a functional assay. When treated with doxorubicin, somatic cells transfected with a genetic construct including the promoter of metaxin and a reporter gene showed increases in expression similar to those observed in fetal oocytes. This demonstrates the direct effect of agent on the regulation of a specific gene.