High adherence is important in microbicide trials, but no adherence interventions to date have demonstrated empiric improvements in microbicide adherence or effectiveness. Approximately midway during the CAPRISA 004 trial, we implemented a novel adherence intervention (Adherence Support Program—ASP), based on an Information–Motivation–Behavioral Skills model and incorporating a Motivational Interviewing approach. We assessed the impact of the ASP on adherence and tenofovir gel effectiveness using a before-and-after comparison. Of the 889 women in the trial, 774 contributed 486.1 women-years of follow-up pre-ASP and 828 contributed 845.7 women-years of follow-up post-ASP. Median adherence rose from 53.6 % pre-ASP to 66.5 % post-ASP. Detectable tenofovir levels increased from 40.6 % pre-ASP to 62.5 % post-ASP in 64 women who had paired tenofovir drug samples. Gel effectiveness improved post-ASP; HIV incidence in the tenofovir gel arm was 24 % lower pre-ASP compared to 47 % lower post-ASP. Following implementation of the ASP, microbicide adherence improved with a concomitant increase in the effectiveness of tenofovir gel.