Spliceosomal introns are important components of eukaryotic genomes, occurring widely within protein-coding genes and being removed through splicing during RNA maturation. Spliceosomal intron removal has long been considered an important foundation of eukaryotic gene expression regulation and cellular function, and has also been proposed as a potential driver of eukaryotic evolution. All extant free-living eukaryotes are known to contain spliceosomal introns. However, whether introns and the spliceosome are essential for the survival of eukaryotic cells remains a fundamental question in biology. In recent years, advances in synthetic biology and genome engineering have provided new strategies for exploring the fundamental components of eukaryotic life. Through synthetic genome design and systematic genome minimization, recent studies have demonstrated that Saccharomyces cerevisiae can survive in the complete absence of all known spliceosomal introns, indicating that spliceosomal introns are not genetic elements essential for yeast viability, and that the spliceosome is likewise not a molecular machine required for yeast survival. This finding challenges the conventional understanding of spliceosomal intron function and spliceosome essentiality, provides a new perspective on the evolutionary significance of introns, and offers guiding principles for the rational design and construction of synthetic eukaryotic genomes. This review systematically summarizes the types and multifaceted functions of introns, with a focus on the construction, viability, and fitness cost of the spliceosomal intron-free Saccharomyces cerevisiae strain SYNE27^α, as well as evidence that the spliceosome is dispensable in this context. It further discusses the implications for the evolutionary retention of introns and the construction of minimal eukaryotic genomes.