To investigate the effect of rare earth element lanthanum on osmotic regulation of navel orange leaves, the two-year-old ‘Newhall’ navel orange trees were used as the test material, and 0 mg/L (CK), 50 mg/L, 150 mg/L and 300 mg/L lanthanum nitrate solutions were sprayed on the navel orange leaves. Subsequently, the young leaves of the middle and upper part of the plant were collected at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h after spraying. Finally, the content of soluble sugar, soluble protein and free proline in the samples were determined. The results showed that the soluble protein content in navel orange leaves (CK) increased first and then decreased at different growth time, while the content of soluble sugar and free proline continued to decrease. The accumulation trend of free proline content in navel orange leaves changed after spraying lanthanum nitrate solution with different concentrations. When the concentration of lanthanum nitrate was 50 mg/L and 150 mg/L, the accumulation of soluble sugar, soluble protein and free proline in navel orange leaves were promoted simultaneously. When the spraying concentration was increased to 300 mg/L, the accumulation of soluble sugar and free proline content in leaves was significantly higher than that of other treatments, but the accumulation of soluble protein content was significantly inhibited. Therefore, it could be concluded that navel orange leaves could adapt to regulate cell osmotic stress by actively accumulating soluble sugar, soluble protein and free proline after spraying lanthanum nitrate. Due to the varieties of osmotic substances, the appropriate concentration that promotes its content accumulation is different, wherein the appropriate concentration of soluble protein content accumulation is ≤ 150 mg/L, and the appropriate concentration of soluble sugar and free proline content accumulation is ≥ 300 mg/L. The results could provide reference for further research on the functional mechanism of rare earth elements on osmotic substances and the regulation of physiological metabolism.