Intensive land-use change, the overgrazing of pastures, and the poor soil management in the Amazon region induce significant soil chemical degradation, causing alterations in the soil phosphorus (P) dynamics. Here, we studied the changes in P fractions and availability throughout the soil profile along a chronosequence composed of four study areas representing the typical land-use transition from forest to pasture for extensive cattle ranching in the Colombian Amazon region: (i) Forest—Deforested—Pasture 4 years old and Pasture established >25 years after deforestation. Soil samples collected at 0–10, 10–20, 20–30, and 30–40 cm depth were used for the sequential fractionation of P, determination of acid phosphatase activity and soil organic carbon (C) content, and calculation of C:organic P (Po) ratio and P stocks. Our results showed that the land-use change caused a decrease of 31.1% in the fractions of labile inorganic P, with the mineralization of organic P by phosphatase enzyme playing an essential role in the P availability. Although according to the C:Po ratio of the deeper layer the P seems to be sufficient to satisfy the plant needs of all the land uses assessed, the exploitation of soil nutrients in pastures reduced by 6.1% the moderately and non-labile P stock. Given the role of cattle ranching in the economy of tropical countries, it is imperative to adopt strategies of soil P management to improve P-use efficiency, avoiding the degradation of grazing land resources while ensuring the long-term sustainability of rangeland livestock and decrease further deforestation of the Amazon rainforest.