Retail stores are a promising setting for improving access to nutritious food. This study opportunistically evaluated an initiative that supported stores in small rural Victorian towns to sell fresh fruit and vegetables. It aimed to measure whether the initiative showed a trend towards improved access to fruit and vegetables in these rural communities. A mixed-methods, pre-post evaluation was employed to measure the range and price of 39 fruits and 45 vegetables, together with 15 interviews with stakeholders 2.5 years after the commencement of the initiative. Twenty-one of 35 eligible stores took up the initiative. Analysis of qualitative and quantitative data showed that the initiative improved the availability of and access to fruit and vegetables, and that stores have a role in improving access to fruit and vegetables. The overall range of fruit and vegetables increased over 18 months from a median of 10 varieties (n = 10) to 17 varieties (n = 15) (P = 0.028) and the prices decreased over 12 months in five out of seven stores where data was available. The capacity to influence availability of fruit and vegetables was affected by time, human resources and community support. Sustaining change to fruit and vegetables access is challenging. Using stores for health promotion may be an effective strategy for improving rural populations’ fruit and vegetable intake.