Early management of spasticity may improve stroke outcome. Botulinum toxin type A (BoNT-A) is recommended treatment for post-stroke spasticity (PSS). However, it is usually administered in the chronic phase of stroke. Our aim was to determine whether the length of time between stroke onset and initial BoNT-A injection has an effect on outcomes after PSS treatment. This multicenter, longitudinal, cohort study included stroke patients (time since onset <12 months) with PSS who received BoNT-A for the first time according to routine practice. The main outcome was the modified Ashworth scale (MAS). Patients were evaluated before BoNT-A injection and then at 4, 12, and 24 weeks of follow-up. Eighty-three patients with PSS were enrolled. MAS showed a significant decrease in PSS at 4 and 12 weeks but not at 24 weeks after treatment. Among the patients with a time between stroke onset and BoNT-A injection >90 days, the MAS were higher at 4 and 12 weeks than at 24 weeks compared to those injected ≤90 days since stroke. Our findings suggest that BoNT-A treatment for PSS should be initiated within 3 months after stroke onset in order to obtain a greater reduction in muscle tone at 1 and 3 months afterwards.