The current study was conducted to explore the ammoniation treatment effects on the chemical composition and in vitro digestibility of rice straw in Chinese Holsteins. For this purpose, rice straw was stored in polyethylene bags (35 × 25 cm, 350 g per bag) including (i) no additives (RS); (ii) 5% urea (5U, dry matter (DM) basis); (iii) 9% corn steep liquor + 5% urea (9C5U, DM basis); (iv) 9C2.5U; and (v) 9C2.5U + 3% molasses (9C2.5U3M, DM basis). The air-dry matter of the mixture was kept at the same level at 55% for all treatments. Fifteen bags (5 treatments × 3 repeats) were prepared and stored at ambient temperature (25 ± 3 °C). The chemical composition and in vitro digestibility were measured at day 60 after storage. Our analysis revealed that all the four ammoniation treatments improved the in vitro DM and neutral detergent fiber (IVNDFD) digestibility. In addition, all the four ammoniation treatments significantly ( < 0.001) increased the levels of crude protein (CP), gas production (GP), acetic acid (AA), butyric acid (BA) and total volatile fatty acid (TVFA) contents of the rice straw and decreased the neutral detergent fiber (NDF) and acid detergent fiber (ADF) of the rice straw compared to the control. Within four treated groups, the 9C5U treatment was most effective. Finally, we concluded that ammoniation treatments increased the nutritive value of rice straw. In addition the 9C5U treatment could be an effective ammoniation treatment for the better utilization of rice straw.