This study examined whether single-gender or mixed-gender PE lessons were more conducive to increasing the physical activity levels of girls. One hundred and twenty girls (12--15 years) wore a wGT3x+ActiGraph accelerometer while participating in basketball games. Time spent in low (LPA), moderate (MPA), vigorous (VPA), and moderate-to-vigorous physical activity (MVPA) were compared between a single-gender and mixed-gender PE lesson. Perceived effort and enjoyment of each lesson were also captured using a three-point scale following the intervention. Girls were found to spend significantly more time in MVPA (p = 0.01) and VPA (p = 0.006) in the single-gender environment. Furthermore, girls spent significantly less time in LPA (p = 0.014). Although girls believed that their effort did not alter with the addition of boys, they did show a preference for taking part in a single-gender environment. Results of this study suggest that segregating PE classes for game-based activities could lead girls to achieve greater health benefits through a higher level of activity.