Cytokinin (CK) inhibits adventitious root (AR) formation in stem cuttings. Little is known, however, about the mechanism underlying the inhibitory effect. In this study, 2 mg l−1 of exogenous 6‐benzyl adenine (6‐BA) was administered to 3 and 7‐day‐old apple rootstocks ‘M.26’ cuttings (3 and 7 days 6‐BA) by transferring them from a rooting medium containing indole‐3‐butanoic acid to the medium containing 6‐BA. Anatomical and morphological observations revealed that the exogenous application of 6‐BA inhibited primordia formation in the 3 days 6‐BA but not the 7 days 6‐BA group. The concentration of auxin (IAA), the ratios of IAA/CK and IAA/abscisic acid were lower in 3 days 6‐BA than in 7 days 6‐BA. Expression analysis of genes known to be associated with AR formation was also analyzed. In the 3 days 6‐BA group, high level of CK inhibited the synthesis and transport of auxin, as a result, low endogenous auxin level suppressed the auxin signaling pathway genes, as were other AR development and cell cycle related genes; all of which had an inhibitory impact on AR primordium formation. On the contrary, low CK level in the 7 days 6‐BA, reduced the inhibitory impact on auxin levels, leading to an upregulated expression of genes known to promote AR primordia formation. Collectively, our data indicated that 3–7 days is the time period in which AR primordia formation occurs in cuttings of ‘M.26’ and that the inhibition of AR development by CK is due to the suppression of AR primordia development over 3–7 days period after culturing in rooting medium.