Maintenance of an anaerobic denitrification system in the obligate human pathogen, Neisseria gonorrhoeae, suggests that an anaerobic lifestyle may be important during the course of infection. Furthermore, mounting evidence suggests that reduction of host-produced nitric oxide has several immunomodulary effects on the host. However, at this point there have been no studies analyzing the complete gonococcal transcriptome response to anaerobiosis. Here we performed deep sequencing to compare the gonococcal transcriptomes of aerobically and anaerobically grown cells. Using the information derived from this sequencing, we discuss the implications of the robust transcriptional response to anaerobic growth. We determined that 198 chromosomal genes were differentially expressed (~10% of the genome) in response to anaerobic conditions. We also observed a large induction of genes encoded within the cryptic plasmid, pJD1. Validation of RNA-seq data using translational-lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. We also confirmed expression and regulation of a small RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family. We also determined that many genes found to be responsive to anaerobiosis have also been shown to be responsive to iron and/or oxidative stress. Gonococci will be subject to many forms of environmental stress, including oxygen-limitation, during the course of infection. Here we determined that the anaerobic stimulon in gonococci was larger than previous studies would suggest. Many new targets for future research have been uncovered, and the results derived from this study may have helped to elucidate factors or mechanisms of virulence that may have otherwise been overlooked.