Start-up packages are a tool for a successful transition to an academic career. This institutional case study examined the faculty experience with start-up packages at one public university in the Southeastern United States, including gender differences, content, negotiation, and perceived outcomes. A mixed-method research design was utilized to answer the study research questions. Data were gathered through an online survey with quantitative and qualitative questions. Data from 121 participants were analyzed using descriptive statistics, chi-square tests of independence, and thematic analysis. Most start-up package agreements included moving expenses, personal computers and software, and start-up funds. Conversely, child daycare, guaranteed junior sabbatical, and salary advancement were the most missing benefits in the agreements. Male faculty obtained, significantly more often than female faculty, a specific number of years for secure funding, laboratory space, and student or postdoc funding in their agreements. Faculty, in general, were not well prepared for the negotiation process and were not aware of what they needed to establish a successful research program. Universities should focus more on the influence of start-up packages on faculty careers because perceived unfair treatment during the negotiation process or administration can influence faculty performance and turnover intentions.