BACKGROUND: In recent years, there was an increased interest in investigating the gender difference in performance and the age of peak performance in ultra-endurance performances such as ultra-triathlon, ultra-running, and ultra-swimming, but not in ultra-cycling. The aim of the present study was to analyze the gender difference in ultra-cycling performance and the age of peak ultra-cycling performance in the 720-km 'Swiss Cycling Marathon', the largest European qualifier for the 'Race Across America'. METHODS: Changes in the cycling speed and age of 985 finishers including 38 women and 947 men competing in the Swiss Cycling Marathon from 2001 to 2012 covering a distance of 720 km with a change of altitude of 4,993 m were analyzed using linear regression. RESULTS: The gender difference in performance was 13.6% for the fastest cyclists ever, 13.9% ± 0.5% for the three fastest cyclists ever and 19.1% ± 3.7% for the ten fastest cyclists ever. The gender difference in performance for the annual top three women and men decreased from 35.0% ± 9.5% in 2001 to 20.4% ± 7.7% in 2012 (r2 = 0.72, p = 0.01). The annual top three women improved cycling speed from 20.3 ± 3.1 km h-1 in 2003 to 24.8 ± 2.4 km h-1 in 2012 (r2 = 0.79, p < 0.01). The cycling speed of the annual top three men remained unchanged at 30.2 ± 0.6 km h-1 (p > 0.05). The age of peak performance for the ten fastest finishers ever was 35.9 ± 9.6 years for men and 38.7 ± 7.8 years for women, respectively (p = 0.47). CONCLUSIONS: The gender difference in ultra-cycling performance decreased over the 2001 to 2012 period in the 720-km Swiss Cycling Marathon for the annual top three cyclists and reached approximately 14%. Both women and men achieved peak performance at the age of approximately 36 to 39 years. Women might close the gender gap in ultra-endurance cycling in longer cycling distances. Future studies need to investigate the gender difference in performance in the Race Across America, the longest nonstop and non-drafting ultra-cycling race in the world.