Abstract Mitochondrial RNA N1‐methyladenosine (m 1 A) is a prevalent and reversible epitranscriptomic modification. While the biological roles of cytosolic m 1 A have been increasingly understood, the causal relationship between site‐specific mitochondrial m 1 A and phenotypic outcomes remain elusive, partly due to the lack of precise editing tools. Here, a CRISPR‐free mitochondrial RNA m 1 A demethylation (MRD) editor is reported, which fuses mitochondria‐localized engineered PUF RNA‐binding protein with the m 1 A demethylase ALKBH3. Independent cellular assays across multiple sites confirm that MRD editor enables precise demethylation of m 1 A in mitochondrial mRNAs and tRNAs, leading to correlated changes in mitochondrial protein levels with minimal off‐target effects. The MRD editor is further employed to systematically investigate how site‐specific mitochondrial m 1 A alterations regulate cell proliferation, ATP production, mitochondrial membrane potential (MMP), and mitochondrial respiration. Finally, in vivo application of the MRD editor reveals that demethylation of m 1 A at the A9 position of mitochondrial tRNA‐Lys (MT‐TK9) induces severe immunodeficiency phenotypes in mice, as evidenced by transcriptomic and histopathological analyses. Collectively, the findings establish MRD as a versatile tool for site‐specific mitochondrial RNA m 1 A editing, offering new insights into the functional dissection of these modifications through chemical biology strategies.