Neisseria gonorrhoeae is one of the most prevalent sexually transmitted infections worldwide. This obligate human pathogen has been extensively studied in vitro, where bacterial factors that are known to contribute to gonococcal disease and their regulation are relatively well defined. However, these in vitro experimental conditions only loosely replicate the host specific environment encountered by the bacteria in vivo. We recently reported on the complete gonococcal transcriptome expressed during natural human mucosal infection using RNA-seq analysis. Gene transcripts expressed in vivo (in vivo expressed factors) included genes encoding antibiotic resistance determinants, and a large number of hypothetical genes. A comparison of the gonococcal transcriptome expressed in vivo with the corresponding strain grown in vitro identified sets of genes regulated by infection, including those regulated by iron and the transcriptional regulatory protein Fur. We highlight here the role of Fur and gonococcal-specific regulatory processes important for infection and pathogenicity. We have determined that the genes controlled by Fur follow the same expression pattern in vivo as described previously in vitro, confirming Fur's regulatory role during infection. Collectively, these studies provide new insights into how bacterial fitness and pathogenicity are modulated during human mucosal infection.