A new generation of a rumen protected lysine product, the third-generation AjiPro-L (A3G, Ajinomoto Co. Inc.), was recently developed. Because the particle size of A3G is smaller than the previous generation product (the second-generation AjiPro-L, A2G), it was expected that A3G has higher intestinal digestibility than A2G due to an increased surface area while the rumen protection of A3G might be compromised. This study was conducted to evaluate the bioavailability of A3G by examining the rumen escape and the fecal excretion of the product. Four dry cows (BW 650–720 kg), fistulated both ruminally and duodenally and fed a corn silage based diet (DMI 13–14 kg/d), were used. Ruminal escape of A2G and A3G was determined by the duodenal digesta collection method with using highly protected l-arginine as a control maker (HP-Arg; Robinson et al., 2011). A2G or A3G was ruminally administrated along with HP-Arg and duodenal digesta was collected every 2 or 3 h for 48–54 h. The digesta was heated with hot water to dissolve fat-containing A2G or A3G particles and homogenized to extract free Lys and Arg. Changes of Lys and Arg concentrations in duodenal digesta were plotted to calculate the area under the curve (AUC). The proportion of AUC of Lys to Arg was defined as the rate of ruminal escape of A3G, based on in situ rumen protection of HP-Arg (>90%). Fecal excretion of A2G or A3G was measured by the 72 h-fecal collection method (Miura et al., 2015). Feces collected individually after ruminal ingestion of the products were homogenized with hot water to extract Lys from A2G or A3G particles in feces. Amounts of Lys excreted in feces were calculated by analyzing free Lys concentration in the extract. Statistical differences were tested by a one-way ANOVA. Rumen escape of A3G (68 ± 7% of initial, mean ± SE) was not different from A2G (73 ± 6%, P = 0.60), but fecal excretion of A3G (18 ± 3%) was significantly lower than that of A2G (32 ± 4%, P < 0.05). These results indicated that A3G can deliver more bioavailable Lys to the absorption site of dairy cows than A2G, thanks to improved intestinal digestibility due to an increased surface area of A3G.