An in vitro experiment was conducted to examine the effect of a protease (papain, EC 3.4.22.2) on ruminal fiber digestibility of alfalfa hay stem. Exactly 100 mg of enzyme powder was solubilized using 5 ml of water. Serum bottles containing 500 mg of alfalfa hay stem, modified McDougall’s buffer and rumen fluid with addition of 0, 50, 100 or 200 µL of the papain solution/g alfalfa hay stem DM were incubated under anaerobic conditions at 39°C for 24 and 48 h. Total gas production and NDF disappearance were determined. Partitioning factor (PF) was calculated as the ratio of NDF degraded (mg) to the volume of gas (mL) produced. Data were analyzed as a completely randomized design by ANOVA using SAS 9.2. The enzyme with a level of 200 µL/g alfalfa hay stem DM significantly (P < 0.05) increased in vitro NDF degradability after 24h incubation (40.2, 41.6, 40.6 and 45.9% in alfalfa hay stem, alfalfa hay stem+50, alfalfa hay stem+100 and alfalfa hay stem+200 µL of enzyme/g alfalfa hay stem DM, respectively). No significant difference was observed after 48h incubation. The PF was also increased significantly (P < 0.01) with addition of all levels of papain (1.57, 1.76, 1.70 and 1.77 in alfalfa hay stem, alfalfa hay stem+50 alfalfa hay stem+100 and alfalfa hay stem+200 µL of enzyme/g alfalfa hay stem DM, respectively). From the result of this experiment, it appears that addition of papain at the highest level has a pronounced effect on the digestion of NDF after 24h incubation. It is concluded that this enzyme has some potential on removing structural proteins in the cell wall, allowing ruminal microbes to gain faster access to digestible substrates.