Ecdysteroids are a class of insect hormones with a characteristic 7 en-6-one moiety in the B-ring, displaying important physiological effects on insects and beneficial pharmacological properties such as decreasing blood cholesterol and glucose levels, anticancer, and wound healing activities. Callisia fragrans (Lindl.) belongs to the Commelinaceae family, native to South America. Previous chemical studies showed the presence of phenolic compounds and amino acids in this plant, and its methanol extract exhibited antiherpetic activity. In this paper, we report the isolation and structure determination of three new and two known ecdysteroids from the stems of C. fragrans. The stems were extracted with hot methanol, suspended in water, and partitioned with n-hexane and chloroform, followed by combined chromatographic separations from the water layer to obtain the compounds. The two known ecdysteroids were identified as 2β,3β,11α,14α-tetrahydroxy-5α-androst-7(8) en-6,17-dione and 2β,3β,14α,17β-tetrahydroxy-5α-androst-7(8)-ene-6-one based on spectral data consistent with literature. The three new compounds (named callecdysterol A, B, and C) had their structures elucidated using HR-ESI-MS and various NMR techniques including 1H, 13C, DEPT, HMBC, COSY, and NOESY, which confirmed their molecular formulas, functional groups, and configurations.