Niche-restricted pathogens are evolutionarily linked with the specific biological fluids that are encountered during infection. Neisseria gonorrhoeae causes the genital infection gonorrhea and is exposed to seminal fluid during sexual transmission. Treatment of N. gonorrhoeae with seminal plasma or purified semen proteins lactoferrin, serum albumin, and prostate-specific antigen each facilitated type IV pilus-mediated twitching motility of the bacterium. Motility in the presence of seminal plasma was characterized by high velocity and low directional persistence. In addition, infection of epithelial cells with N. gonorrhoeae in the presence of seminal plasma resulted in enhanced microcolony formation. Close association of multiple pili in the form of bundles was also disrupted after seminal plasma treatment leading to an increase in the number of single pilus filaments on the bacterial surface. Thus, exposure of N. gonorrhoeae to seminal plasma is proposed to alter bacterial motility and aggregation characteristics to influence the processes of transmission and colonization. IMPORTANCE There are greater than 100 million estimated new cases of gonorrhea annually worldwide. Research characterizing the mechanisms of pathogenesis and transmission of Neisseria gonorrhoeae is important for developing new prevention strategies, since antibiotic resistance of the organism is becoming increasingly prevalent. Our work identifies seminal plasma as a mediator of N. gonorrhoeae twitching motility and microcolony formation through functional modification of the type IV pilus. These findings provide insight into motility dynamics and epithelial cell colonization under conditions that are relevant to sexual transmission. Type IV pili are common virulence factors with diverse functions among bacterial pathogens, and this work identifies interactions between type IV pili and the host environment. Finally, this work illustrates the importance of the host environment and niche-specific fluids on microbial pathogenesis.