The long 5′ untranslated region (5′UTR) exhibits enhancer activity in translation of rice OsMac3 mRNA. In this report, we describe elements of OsMac3 5′UTR that may be responsible for its enhancer activity, including a long uORF and several secondary structure elements. OsMac3 5′UTR can be dissected into three stem-loop structures SL1, small SL and SL2, where the uORF starts within SL1 and ends within SL2. As expected, uORF inhibits translation of downstream ORF since deletion of the uORF AUG or the SL1 stem-loop increases translation by approximately two-fold. Thus, the 158 nt 3′ region of the 5′UTR lacking SL1 together with the AUG uORF, which has significant enhancer activity, was named dMac3. We investigated two critical regions within dMac3 mRNA that influence its translation: SL2, which destabilization potentially decreases translation activity, and another 13 nt located downstream of SL2. We further confirmed that dMac3 promotes mRNA translation initiation in an in vitro translation system and during transient expression in either cultured cells or Nicotiana benthamiana leaves. Thus, the dMac3 5′UTR is a useful tool for efficient protein production in various in vitro and in vivo translation systems.