The environmental consequences of nano-CuO particles have gained significant attention in recent decades. Identification of the mechanisms for soil and plant responses with respect to the chemical speciation of nano-CuO (mainly the exchangeable and reducible fractions) remains scarce. Here, we analyzed different chemical speciation of Cu and DTPA-extractable Cu over 42 days in (1) control soil without Cu addition; (2) soil treated with nano-CuO particles; and (3) soil treated with CuSO solution. The applied dose was 500 mg Cu kg and maize was grown in these soils. Plant growth was inhibited, but the inhibition by nano-CuO was slightly weaker compared to CuSO. Cu accumulations were similar in the roots for CuSO and nano-CuO treatments, but significantly higher in the shoots for CuSO treatment. This indicates that Cu from nano-CuO-treated soils mainly accumulated in roots but rarely transferred to shoots. Enzyme activities on the rhizoplane visualized by zymography were strongly depressed by CuSO but slightly inhibited by nano-CuO. Microbial community diversity measured by 16S rRNA was the lowest in CuSO-treated soils among three treatments. These results were explained by the following mechanisms: (1) Gradual increases of DTPA-extractable and exchangeable Cu were found in nano-CuO-treated soil, and the final concentrations at day 42 were only half of those in CuSO-treated soil; (2) Enzyme activities on the rhizoplane were positively related to soil pH and negatively correlated with DTPA-extractable and exchangeable Cu; (3) Even though reducible Cu in nano-CuO-treated soils was 1.3 times higher than in CuSO-treated soils, indicating stronger nano-accrued oxidative stress in nano-CuO-treated soils, the toxicity induced by nano-CuO particles was still weaker than CuSO. Nevertheless, the toxicity of Cu particles to plants and microbes mainly depends on the gradually-released bioavailable Cu. This demonstrates the greater importance of bioavailable Cu concentrations for toxicity modulation rather than the scale of Cu particles.