Objective To compare the impact of different central nervous system (CNS) prophylaxis regimens and the timing of prophylaxis on the outcome of patients with first-treatment for diffuse large B-cell lymphoma (DLBCL). Methods A retrospective case series was conducted on patients diagnosed with primary DLBCL at the Hunan Cancer Hospital between November 2009 and February 2022. The patients were divided into two groups: a CNS prophylaxis group and a non-prophylaxis group. The CNS prophylaxis group was then divided into three groups according to their prophylaxis regimens: IT-MTX, IV HD-MTX and IT-MTX plus IV HD-MTX. The CNS relapse-free survival (CRFS) of the prophylaxis group and the non-prophylaxis group was evaluated. Furthermore, the impact of varying prophylaxis regimens on overall survival (OS) and progression-free survival (PFS) rates was analysed, and the influence of different injection timing of HD-MTX on OS and PFS rates of patients was compared. Additionally, the effect of different injection timing of MTX on patients' OS and PFS rates was investigated. OS, PFS and CRFS in the population of patients rated at intermediate to high risk of CNS replase based on the CNS-IPI score were also analyzed. Results A total of 301 patients diagnosed with primary DLBCL were treated in the study. Among these patients, 152 (50.50%) underwent CNS prophylaxis, while 149 (49.50%) did not. The median follow-up duration was 66.90 (1.40~166.73) months, with an overall patient 5-year OS rate of 39.87% and a 5-year PFS rate of 31.89%. The 5-year OS and PFS rates were 59.50% and 61.89% respectively in the CNS prevention group, and 63.05% and 67.08% respectively in the non-prevention group. There was no statistically significant difference in total OS and total PFS between the two groups ( P=0.615, P=0.821). However, a significant difference was found in CRFS between the two groups ( HR=0.205, 95% CI: 0.099~0.425, P<0.000 1). In the CNS prophylaxis group, the number of patients was recorded as follows: 83 in the IT-MTX group, 46 in the IV HD-MTX group and 23 in the IT-MTX combined with IV HD-MTX group. No statistically significant difference was observed in either OS ( P=0.557) or PFS ( P=0.455) between the three groups. The analysis of the timing of HD-MTX injection revealed that the 5-year OS and PFS rates were 74.54% and 79.39% for patients in the chemo-interval administration group, and 55.05% and 46.63% for patients in the post-chemo administration group. The OS ( P=0.012) and PFS ( P=0.006) in the chemo-interval administration group were superior to those in the post-chemo administration group. According to the CNS-IPI score, there were 136 cases of intermediate-high risk patients, of which 76 cases (55.88%) had CNS prophylaxis and 60 cases (44.12%) did not have CNS prophylaxis. The median OS for the former group was 71.33 months, with a 5-year OS rate of 53.95%, while the latter group exhibited a median OS of 52.73 months and a 5-year OS rate of 46.46%. A statistical analysis revealed no significant differences between the two groups( P=0.851). The 5-year PFS rates of the two groups were 60.25% and 44.79%, respectively, and there was a significant difference ( P=0.015). There were 15 cases of CNS relapse in the group without CNS prophylaxis but no CNS recurrence in the group with CNS prophylaxis, and there was a significant difference in CRFS between the two groups ( P<0.000 1). Conclusion There was no significant difference in the survival impact of the three prophylactic strategies examined in patients with DLBCL, but HD-MTX administered between chemotherapy cycles prolonged the PFS of paients along with a modest survival benefit compared with post-chemotherapy administration, although no significant increase in survival was observed. CNS prophylaxis with HD-MTX in DLBCL patients at intermediate to high risk of CNS replase has been shown to significantly reduce the risk of CNS replase and prolong the PFS of patients.