This study aimed to assess associations between school HIV education and protective sexual behaviors and sexually transmitted disease (STD)/HIV diagnosis with a representative sample of male and female high school students. Data from male and female adolescent participants in the 1999, 2001 and 2003 Massachusetts Youth Risk Behavior Survey (n = 12,243) were analyzed. Adjusted regression analyses stratified by gender were conducted to assess relationships between school HIV education exposure and the following outcomes: no sexual initiation, condom use at last sex, no multiple sex partners in the past three months and no history of STD/HIV diagnosis. Participants were mostly White (75%) and were 51% male; the subsample of sexually active students was younger than the total sample but was otherwise similar in demographics. School HIV education was reported by 93% of our sample and was significantly related to sexual initiation among boys (odds ratio = 1.9, 95% confidence interval = 1.4-2.7) but not girls. Among sexually experienced students (n = 4752), boys reporting exposure to school HIV education were significantly more likely to report condom use (odds ratio = 2.2, 95% confidence interval = 1.6-3.1), no multiple sex partners (odds ratio = 3.2, 95% confidence interval = 2.3-4.4) and no STD/HIV diagnosis (odds ratio = 3.2, 95% confidence interval = 2.0-5.0); girls reporting such exposure were significantly more likely to report no multiple sex partners (odds ratio = 2.2, 95% confidence interval = 1.3-3.6). In conclusion, exposure to school HIV education is associated with sexual protective behaviors and reduced likelihood of STD/HIV diagnosis for boys but less so for girls, suggesting the need for more gender-tailored approaches to school HIV education. (Contains 2 tables.)