SUMMARYThe protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we used RNA immunoprecipitation followed by high‐throughput sequencing (RIP‐Seq) to identify MatK's preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for splice site selection, and we discovered alternative 5′‐splice sites in three MatK target introns. The resulting alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA‐K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and a trnK‐matK over‐expressor exhibited elevated levels of the spliced tRNA‐K(UUU). Ribosome profiling analysis of the overexpressor revealed a significant up‐shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA‐K(UUU), enabling the differential expression of functional chloroplast gene groups.