Context. Reducing the environmental impact of animal production is becoming a really hot topic, especially with raised concerns over excessive flows of nitrogen and phosphorus (P) to the environment. Aims. The present study was conducted to determine the effects of phytase supplementation on growth, plasma biochemistry, bone mineralisation and P utilisation of pre-lay pullets fed varying levels of non-phytate P. Methods. A total of 240 Lohmann pullet chicks were randomly allocated to one of six dietary treatments with eight replicate cages (5 birds per cage) per treatment. Six treatments included three phytase-free diets and three diets supplemented with 1000 U/kg phytase; the non-phytate P levels were 2.75-2.50-2.25, 3.75-3.50-3.25 and 4.75-4.50-4.25 g/kg in the former, and 1.75-1.50-1.25, 2.75-2.50-2.25 and 3.75-3.50-3.25 g/kg in the latter, for the age of 0-4, 4-8 and 8-16 weeks respectively. Key results. No significant differences were found for growth performance, plasma biochemistry (calcium, P, alkaline phosphatase and albumin) and bone mineralisation among dietary treatments, but P retention (%) was different (P < 0.001). Analysis of planned contrasts showed that phytase supplementation increased phytate P retention (P < 0.001), and improving the utilisation of phytate P tended most efficiently under low P conditions. Total P retention rate was reduced slightly by phytase supplementation (P < 0.05). Conclusions. The results indicated that dietary non-phytate P level could possibly be reduced to 1.75, 1.50 and 1.25 g/kg for 0-4, 4-8 and 8-16 weeks of age respectively after phytase supplementation, without compromising pullet growth and performance during the pre-laying period. Implications. The results of this study will contribute to decreasing P excretion by poultry and reducing the potential environmental impact with land application of manure.