Fruiting branches and floral buds are forming well before squares are visible and determine cotton (Gossypium hirsutum L.) productivity. Pre-soaking with plant growth regulators (PGRs) affects the quantity of floral buds. However， studies illustrating the physiological mechanism of floral bud differentiation in response to PGRs are lacking. To address this， cotton seeds were primed with water (control)， 5 mg L gibberellic acid (GA)， 25 mg L N6-benzyladenine (6-BA)， and 150 mg L dimethyl piperidinium chloride (DPC) respectively. Results showed that plants from seed pre-treated with GA and 6-BA differentiated more floral buds relative to control， while DPC application initiated less floral buds than control. GA and 6-BA application significantly increased the levels of zeatin riboside (ZR) by up-regulating IPT expression and gibberellic acid (GA) but decreased the indole-3-acetic acid (IAA) content. Consequently， the ZR/IAA and GA/IAA ratios were markedly increased， contributing to higher floral bud numbers. Contrasting results were observed for DPC treatment. Additionally， GA and 6-BA treatments up-regulated GhSOC1， GhMADS13 and GhAGL24 expression， which was associated with higher sucrose contents mainly attributed to higher endogenous ZR levels， inducing floral initiation. Whereas the GhMADS13 was down-regulated to suppress floral bud differentiation under DPC application. Surprisingly， the floral-associated genes were more sensitive to GA than 6-BA， which induced the differences in bud numbers at the beginning of flower bud differentiation. Thus， we conclude that seed pre-treated with PGRs affected hormone content， induced sugar accumulation in apical buds and regulated genes involved in floral induction， which impacted floral bud differentiation.