The columnar growth trait of apple (Malus×domesticaBorkh.) is genetically controlled by the Columnar (Co) locus on 10 chromosomes, including several candidate genes. Except forMdCo31, other candidate genes at theColocus are less elucidated. In this study, a strategy of step-by-step screening was adopted to select 11 candidate genes by experimental cloning, transient expression, and genetic transformation. There existed several SNPs in four genes by sequence alignment in columnar and non-columnar apples. Two genes were detected in the nucleus and three genes in the cell membrane, other genes were located in multiple cellular structures by subcellular location. Ectopic expression demonstrated that more branching occurred inMdCo38-OE by upregulatingNtPIN1andNtGA2oxand enlarged leaves inMdCo41-OE tobaccos by upregulatingNtCCDs. Transcripts ofMdCo38andMdCo41were associated with theCogenotypes in apples. The results indicate thatMdCo38andMdCo41are involved in the columnar growth phenotype in apple, probably through altering polar auxin transport, active gibberellin levels, and strigolactone biosynthesis.