Microsomal triglyceride transfer protein (MTTP) is an essential chaperone that assists in the assembly of apolipoprotein B-containing lipoproteins to transport lipids. We have shown that microRNA (miR)-30c regulates MTTP expression but other members of the same family do not. Further, we showed that interactions between miR-30c seed sequence and the 3΄-untranslated region (UTR) of the MTTP mRNA are critical for this regulation. The same seed sequence is shared by all the members of the miR-30 family. Therefore, it is unclear why only miR-30c regulates MTTP expression. Bioinformatics analysis revealed that, miR-30c interacts with MTTP mRNA involving supplementary site, besides seed sequence, forming an intervening loop. Here, we evaluated the importance of the supplementary site and the size of the intervening loop in miR-30c/MTTP mRNA interactions by cloning MTTP 3΄-UTR at the end of the luciferase gene and subjecting it to site-directed mutagenesis. Reducing the number of base pairs at the supplementary site abolished the ability of miR-30c to reduce luciferase activity. However, increasing the number of base pairs at the supplementary site, seed sequence or in the intervening loop enhanced the efficacy of miR-30c in reducing luciferase activity. These studies demonstrated that the supplementary site of miR-30c is, but the intervening loop is not, critical for binding to the MTTP mRNA. To our knowledge, this is the first demonstration that miRs might require both seed and supplementary interactions to regulate target mRNA specificity. Further, this study suggests that more potent miR-30c mimics could be synthesized by increasing base pairing in the loop region.