Strigolactones (SLs) are a well-known class of plant hormones, which are involved in a number of developmental and adaptation processes and mediate different interspecific interactions. In spite of the growing knowledge on SL biosynthesis and signal transduction, effects of structural modifications on the activity and efficiency of SLs and their analogs remain largely elusive. SLs are characterized by the presence of a lactone ring (D-ring) that is connected by an enol ether bridge to a second moiety. In this study, we investigated the effect of additional D-ring methylation of SL analogs on their transcription regulating activity. For this purpose, we compared the SL analogs MP13 and AR8, which differ only by the presence of a methyl group at the C-3ʹ atom in the latter. Transcription regulating activity was determined by quantitative real-time PCR measurement of transcript levels of SL-dependent, feed-back regulated genes in treated wild type and ccd7 mutant rice seedlings. Results obtained indicate that C-3ʹ methylation reduces the transcription regulating activity, as shown by the more pronounced suppression of the SL biosynthesis genes DWARF27 (D27) and CAROTENOID CLEAVAGE DIOXYGENASES (CCD7 and CCD8) and higher induction of the SL signaling repressor gene DWARF53 (D53) in MP13 treated seedlings. These results are consistent with a recent study on the biological activities of MP13 and AR8.