Purpose <p>Phosphorus (P) has long been recognized to be present in humic substances. However, the responses of P forms and availability in humic substance fractions to different fertilization practices remain unclear. This study aimed to investigate the influence of mineral and organic fertilization on the forms and availability of P in humic acid and humin in a Mollisol through a field experiment.</p> Methods <p>This experiment included six treatments: control without fertilizers, mineral nitrogen, phosphorus, and potassium fertilizers (NPK), pig manure applied at 30,000 (M1) and 60,000&#xa0;kg ha^–1 (M2), and M1 or M2 combined with NPK. Solution ³¹P nuclear magnetic resonance and enzymatic hydrolysis techniques were used to analyze the forms and availability of P.</p> Results <p>The P associated with humic substance fractions was predominant in organic forms, mostly orthophosphate monoesters. Humic acid and humin contained 42.4–54.3% and 40.2–54.2% of labile inorganic P and 9.61–13.4% and 9.45–15.2% of labile organic P, respectively. The concentrations of total P and P forms were higher in humic acid than in humin. Among different fertilization treatments, the concentrations of total P and P forms were generally higher in treatments with high than with low manure rates, with than without manure, and with than without mineral fertilizers.</p> Conclusions <p>High rate manure combined with mineral fertilizers was more effective in enhancing the availability of P associated with humic substance fractions. Our findings can help improve P management for more efficient crop uptake by enhancing our knowledge of P forms and availability.</p>