Adolescence is a time of emotional, physical and psychological development and contributes substantially to the well being of the individual in adulthood. Consequently, understanding their social and reproductive behaviour is of tremendous policy importance. This study was designed to investigate HIV knowledge and safety sexual practices among adolescent girls in Benin-city, Edo State, Nigeria. Methods: A cross sectional study was conducted among 100 randomly selected female students in SS1 and 2 from two secondary schools in Benin-city. A semi structured questionnaire was self administered to obtain information on respondents’ socio-demographic characteristics, knowledge of HIV and safety practices. A 10-point knowledge scale graded, <5 and ≥6 as poor and good knowledge respectively, was used to measure knowledge of HIV; while a 5-point practice scale graded <2 and ≥3 was used as unsatisfactory and satisfactory safety sexual practices on HIV prevention respectively. Descriptive statistics, chi-square test and logistic regression were used to analyse the data with level of significance set at 0.05. Results: The mean age of respondents was 12.70 ± 1.2 years, 50.0% were in SS2 and 65.0% had been in Benin-city from birth. The overall mean knowledge score of respondents was 14.70 ± 4.43 with 60.0%, 20.0%, 40.0% and 70.0% having good knowledge on perinatal/vertical transmission, sexual and parenteral transmission, epidemiological factors and preventive measures respectively. Misconceptions about HIV transmission include: HIV cannot be transmitted through oral sex, HIV can be transmitted by mosquitoes and naked eyes can detect who is infected. In addition, there was a significant difference between class of respondents and their general knowledge of HIV (P < 0.05). Respondents in SS2 were more likely to have good knowledge of HIV than their SS1 counterpart (OR = 3.43 95% CI = 1.461 - 8.057). Furthermore, respondents whose mothers attained at least secondary school are more likely to exhibit satisfactory safety sexual practices on HIV prevention than their counterpart whose mothers had no formal education (OR = 2.67 95% CI = 0.619 - 11.493). Conclusions: There were knowledge deficiencies in sexual and parenteral transmission, epidemiological factors of HIV as well as some misconceptions about the transmission of the HIV virus. Therefore, more awareness and health education interventions are needed at the post primary level to curb the spread of the virus.