With only a small percentage of the population engaging in regular physical activity (PA), it is important to determine what barriers are related to PA behavior. Further, because the majority of adults work outside the home, worksites may be an important channel for reaching a large percentage of the adult population with health promotion programs. Therefore, the purpose of this study was to qualitatively identify perceived barriers reported by participants in a worksite-based PA program. Email invitations to participate in focus groups were sent to a random sample of potential participants from a large city government in the intermountain western USA. Female and male respondents were assigned to a focus group with other participants of the same gender. During the focus groups, participants were asked open-ended questions configured from barriers to PA participation commonly reported in the literature. Responses were transcribed verbatim from a digital audio recorder and each transcript was analyzed using the qualitative data analysis program QSR NUD*IST version N6. Open coding was used to categorize and label similar data into themes. Axial coding was used to relate similar categories to the central phenomena of interest (i.e., barriers to PA participation). Sixty-nine employees (age: 44.97 ± 10.47 years, 88% Caucasian) were recruited to participate in 12 focus groups. Overall, females (n = 25, 6 focus groups, age = 44.92 ± 10.12 years, 88 % Caucasian) and males (n = 44, 6 focus groups, age = 45 ± 10.42 years, 88.6 % Caucasian) indicated financial constraints as a concern when deciding to participate in PA. Other common barriers relating to PA participation included lack of time and lack of motivation. While these were commonly reported barriers, males and females responded differently. Females were more likely to identify child care as an issue related to time, and weather conditions related to lack of motivation. Conversely, males were more likely to identify lack of convenience as an issue related to time, and physical fatigue related to lack of motivation. To date, few studies have qualitatively explored how gender differences may impact the identification of barriers to PA participation. Our findings provide further support for the need of using models in which gender is considered a factor between perceived barriers and PA participation. Future research should focus on gender specific factors that influence participation in PA. Keyword(s): adult physical activity/fitness, health promotion, worksite