1. The objective of this study was to test the effect of microbial phytase and calcium (Ca) levels on the Zn bioavailability depending on the Zn source. 2. A total of 144, one-day-old broilers received a diet containing 40 mg Zn/kg for 1 week. They were then assigned to one of the eight experimental treatments during 2 weeks. Diets contained 27 mg native Zn and were formulated according to a 2 × 2 × 2 factorial design with two added Zn sources (23 mg Zn/kg), sulphate (ZnSO4) or oxide (ZnO); two dietary Ca levels of either 6 (low) or 10 g/kg (moderate); and two microbial phytase levels, of either 0 or 750 phytase unit/kg. 3. Moderate Ca plus phytase improved body weight gain (BWG) and low Ca level without microbial phytase decreased tibia weight (Ca × Phy; p = 0.049). Zinc oxide resulted in a higher tibia development (i.e. tibia weight and length; p < 0.05) and BWG (p = 0.009) and lower FCR (p < 0.001) compared to ZnSO4. 4. Phytase improved tibia Zn concentration especially in birds given ZnSO4 compared to ZnO (Zn × Phy; p = 0.049). Moderate Ca diets improved tibial characteristics and Zn deposition compared to low Ca diets (p < 0.05). 5. Soluble Zn in the gizzard increased in the presence of phytase (p = 0.011), while higher dietary Ca reduced this in birds receiving ZnO (p = 0.004). In the jejunum, Zn solubility was higher with phytase (p = 0.008). 6. Under the conditions of this study, dietary levels of Ca and microbial phytase affect Zn availability in broilers more than the Zn source.