An investigation was conducted to study the effect of humic substance (HS) on the phosphorus (P) solubility in acidic soil. The soil (2.5 g), HS (0, 0.5, and 2.5 g), and P as monocalcium phosphate (0.31 and 1.25 g P kg-1 soil) were mixed with 50 mL distilled water and two different sequences of adding HS and P were used. The results indicated that the P concentration in water and 0.01 mol L-1 CaCl2 solution increased with increasing amounts of humic substance. The concentrations of Fe and Al were also increased. However, Olsen P decreased with increasing amount of humic substance.Water-soluble P concentrations from P rates at 0.31 and 1.25 g P kg-1 soil in the treatment with 0.5 g (2.5 g) humic substance addition were 360% and 70% (500% and 90%) higher, respectively, than those in the treatment with no humic substance addition. P extracted by 0.01 mol L-1 CaCl2 in the treatments with 0.5 and 2.5 g humic substance addition was increased by 400% and 540%, respectively, compared with that in the treatment without humic substance at the rate of 0.31 g P kg-1 soil, while the corresponding P concentrations were increased by 80% and 90% at the rate of 1.25 g P kg-1 soil. The order of mixing humic substance and phosphate did not significantly affect desorbed P and labile P extracted with CaCl2.