BACKGROUND: In Malawi, childhood pneumococcal conjugate vaccination has not achieved expected herd protection despite the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in 2011. Vaccine efficacy and longevity of protection are crucial in achieving herd protection. We previously found that PCV13 offers immediate protection from experimental pneumococcal carriage in Malawian adults, but of uncertain duration. In this follow-up study, we aimed to evaluate the longevity of protection of PCV13 vaccination against experimental human pneumococcal carriage measured by 1-year post-vaccination homotypic Streptococcus pneumoniae serotype 6B (Spn6B) rechallenge in healthy Malawian adults. METHODS: We previously conducted a double-blind, parallel-arm, randomised controlled trial investigating the efficacy of PCV13 or placebo against Spn6B carriage in healthy adults (aged 18-40 years) in Blantyre, Malawi. This longitudinal follow-up study re-recruited all eligible participants available at 1 year post-vaccination. Eligibility criteria for participation remained the same as those for the initial trial. Individuals with safety concerns, including those at increased risk of invasive pneumococcal disease or with close contacts at risk, were excluded from study participation. Participants with natural carriage other than Spn6B were included in the study. Participants were rechallenged intranasally with 80 000 colony-forming units of Spn6B per naris. The primary endpoint was experimental pneumococcal carriage, established by culture of nasal wash samples collected at days 2, 7, and 14 after rechallenge, and was assessed in all participants who received the rechallenge inoculation. The original trial was registered with the Pan African Clinical Trial Registry (PACTR202008503507113) and is closed. FINDINGS: Participants were re-recruited from May 10, 2022, to Aug 22, 2023. 137 participants (77% male; median age 26 years [IQR 23-29]) were re-recruited (57 PCV13 and 80 placebo) and completed the study protocol. In the log-binomial adjusted model, vaccine protection on the probability of Spn6B carriage following rechallenge was 73% (relative risk [RR] 0·27, 95% CI 0·08-0·98, p=0·047). Previous experimental Spn6B carriage reduced the vaccine protective effect (RR 2·20, 0·85-5·67, p=0·10). The effect of PCV13 vaccination was reduced within previous Spn6B carriers (RR 8·95, 2·27-35·33, p=0·002) and previous natural pneumococcal carriers (RR 14·31, 1·65-124·20, p=0·016). INTERPRETATION: The study indicates that PCV13 vaccination has a role in preventing pneumococcal carriage for at least 1 year, showing strong interactions with natural carriage events. These findings suggest that the inability to achieve herd protection in Malawi is probably due to factors other than the efficacy or duration of protection of the PCV13 vaccine. FUNDING: Wellcome Trust.