Background: We aimed to evaluate the effect of the number of neoadjuvant chemotherapy (NAC) cycles and the adding adjuvant chemotherapy (AC) after NAC in muscle-invasive bladder cancer (MIBC) on overall survival (OS). Methods: This multicenter retrospective study included 2674 patients with MIBC who underwent radical cystectomy (RC) from 36 institutions within the Japanese Urological Oncology Group. Among them, we selected 1687 patients with cT2-4NxM0 who were treated with RC alone or RC plus perioperative chemotherapy. We compared the effect of the number of NAC cycles (2 vs. ≥3 cycles) and the addition of AC on OS. Cox proportional-hazards regression was used to assess the association of treatment received with OS. Results: Of 1687 patients, 946 were treated with NAC with a median of 3 cycles. Use of NAC significantly prolonged OS compared to the RC alone. The pathological complete response rate was not significantly different between the 2 cycles (22.9%) and ≥3 cycles (27.5%, P = 0.112) groups. OS was not significantly different between the groups (P = 0.559). Multivariable Cox regression analysis showed that pathological high-risk (ypT2–4, pT3–4, or pN+) or cisplatin ineligibility were significantly associated with poor OS, but not the number of NAC cycles (P = 0.238). We identified 942 pathologically high-risk patients after RC who were eligible for AC. We observed no significant OS improvement with addition of AC after NAC as intensive perioperative chemotherapy. The primary limitation is selection bias from confounding by clinical indication. Conclusions: The impact of 3 or more NAC cycles and the addition of AC on OS in MIBC patients treated with RC may be limited.