To measure trends in HIV incidence and serial prevalence by sex in a intravenous drug users (IDUs) and heterosexuals (HT) cohort recruited in a counselling centre in Valencia (1988-2005). Serial prevalence and incidence rates were calculated and modelled by logistic and Poisson regression respectively. 5948 IDUs and 13343 HT were recruited. Prevalence was higher among female IDUs (46% vs. 41%), and female HT (4.1% vs. 2%). For IDUs, an interaction (P = 0.005) between sex and calendar was detected. Age-adjusted prevalence showed faster yearly decline in men (OR = 0.87 95%CI: 0.85-0.88) than in women (OR = 0.91 95%CI: 0.88-0.93). Incidence was higher in female IDUs (9.79% p-y) than in men, (5.38% p-y) with an annual decrease for both of 11%. HIV incidence was higher in female HT (0.62% p-y) compared to men 0.23% p-y with a 21% yearly decline. Gender differences in HIV prevalence and incidence trends have been detected. Women showed an increased vulnerability to infection in a country whose HIV epidemic has been largely driven by IDUs.