The characteristics of microbial glycogen have not been well described, but have implications for its analysis and digestion characteristics. A series of analyses, and comparisons carried out as randomized complete block designs were performed on glycogen isolated from protozoa from ruminal inoculum incubated in vitro with glucose. The isolated protozoal glycogen (IPG) was in the form of water and 0.9% NaCl insoluble granules 1.3 to 1.9 mm in length. IPG was not obviously birefringent under polarized light. α-Glucan was measured by AOAC Official Method 2014.10 as detected glucose × 0.9. α-Glucan contents of beef glycogen, wheat starch (WS), corn starch (CS), and IPG on a dry matter (DM) basis were 98.1, 99.5, 100.0, and 98.3%, respectively. Duplicate samples (each 50 mg in 5 mL water) were incubated at successively increasing temperatures (22, 37, 50, 57, 65, 70, 75, 80, and 85°C) for 30 min with vortexing every 5 min for 5 s. Gelatinization temperature was determined as the point at which transmittance % at 650 nm departed from baseline measurements of unheated samples. WS, CS, and IPG had gelatinization temperatures of 57, 65, and 65°C, respectively. Subsequent enzymatic hydrolysis of 0.2 mL of the sample suspensions was performed with addition of 1 mL 0.1 M Na acetate buffer (pH 5.0), and 4 U amyloglucosidase incubated for 2 h at 39°C. Digested α-glucan as a percentage of initial CS, WS, and IPG were 8.3, 9.0, and 24.7% for ungelatinized vs. 100.0, 88.9, and 95.6% for gelatinized (85°C) samples, respectively. Effects of sample, gelatinization, and sample × gelatinization all were P < 0.01 (standard error of the difference; SED = 0.4). Incubation of ungelatinized suspensions of IPG for 24 h at 57°C with 0, 1.8, or 4 U amyloglucosidase with Na acetate buffer gave α-glucan values of 0.03, 14.3, and 15.0% of DM, respectively (P < 0.01, SED = 0.06). Similar to native starch, gelatinization was required to achieve more extensive enzymatic hydrolysis of IPG. This suggests that protozoal glycogen digestion characteristics may be similar to those of native starch, and that IPG requires gelatinization for analysis involving enzymatic hydrolysis.