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Both zinc (Zn) and iron (Fe) are essential micro-nutrients for plant growth and development, yet their levels in plants are tightly regulated to prevent either deficiency or phytotoxicity. In agronomic reality, such an imbalance of metal bioavailability to crops occurs frequently. Thus, mining genetic resources to improve crop traits relevant to metal homeostasis is a great challenge to ensure crop yield and food quality. This study functionally identified an uncharacterized metallochaperone family HIPP protein gene <i>Heavy Metal Associated Isoprenylated Plant Proteins 33</i> (<i>OsHIPP33</i>) in rice (<i>Oryza sativa</i>). <i>OsHIPP33</i> resides in the nucleus and plasma membrane and constitutively expresses throughout the lifespan. Transcription of <i>OsHIPP33</i> is not induced by deprivation of Zn and Fe but upregulated under excessive Zn and Fe stress. In a short-term (one month) hydroponic study with the normal Zn and Fe supply, there were no significant changes in the growth and metal accumulation between the knockout (<i>OsHIPP33</i>) or knockdown (RNA interference) mutant lines and wild-type, while the long-term field trials (for two successive years) demonstrated that the mutation of <i>OsHIPP33</i> significantly compromised the rice growth and development (such as rice leave tissues, panicle length, spikelet fertility, seed weight per plant, 1000-grain weight, etc.), with the mature grain yield of <i>OsHIPP33</i> and RNAi lines reduced by 52% and 12–15% respectively, compared with wild-type. Furthermore, the accumulation of Zn and Fe in rice straw, husk and brown rice was also reduced. These results suggest that the disruption of <i>OsHIPP33</i> can dampen rice agronomic traits, signifying that <i>OsHIPP33</i> expression is required for Zn and Fe homeostasis and subsequent production of rice grains.