Cross-pollination affects the fruit characteristics of many crops but the effects of cross-pollination on fruit quality of strawberry (Fragaria × ananassa Duch.) are poorly known. This study determined how cross-pollination affects fruit quality of the strawberry cultivar, Redlands Joy, under controlled environment conditions. Plants were allocated to one of four treatments, with all flowers on each plant receiving either: (1) unassisted self-pollination (Autogamy); (2) hand-pollination with Redlands Joy pollen (Self); (3) hand-pollination with cross-pollen from a small-fruited cultivar (Sugarbaby); or (4) hand-pollination with cross-pollen from a large-fruited cultivar (Rubygem). Cross-pollination did not significantly affect plant yield or fruit mass, size, shape, firmness or shelf life. However, cross-pollination affected fruit colour and taste attributes. Cross-pollinated fruit were 3%–5% darker than self-pollinated fruit. They also had 26%–34% lower acidity and 43%–58% higher Brix:acid ratio. Cross-pollination by Sugarbaby increased fruit P, K, Ca, Fe and Mn, but decreased B, Cu and Zn, concentrations. Cross-pollination by Rubygem increased fruit Mn, but decreased K and Na, concentrations and reduced shelf life. Fruit mass, length, diameter and firmness within all treatments increased with increasing numbers of fertilized seeds per fruit. Hand self-pollinated fruit had a higher percentage of fertilized seeds than fruit arising from autogamy and they were also darker, redder, firmer, and had a longer shelf life, higher protein concentration, and lower Al and Na concentrations. The results indicate that strawberry fruit quality can be affected by both the source of pollen and the number of stigmas pollinated.