Plasmodium falciparum gametocytes are usually present in peripheral blood at a very low level, thus requiring a sensitive assay detection method. In this study, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed for clinical detection of P. falciparum gametocytes. Transcripts of Pfs16 of sexually committed ring and Pfs25 of mature gametocytes were detected by RT-LAMP in 82 clinical blood samples using nested RT-PCR as a gold standard. RT-LAMP demonstrated a detection limit of 1 parasitized red blood cell (RBC)/500microl of blood for both Pfs16 and Pfs25 transcripts. For Pfs16 transcript, RT-LAMP detected all 30 samples positive by nested RT-PCR (100% sensitivity) and 1 in 52 samples negative by nested RT-PCR (98.1% specificity). For Pfs25 transcript, RT-LAMP detected all 15 samples positive by nested RT-PCR (100% sensitivity) and none of 67 samples negative by nested RT-PCR (100% specificity). Negative predictive value (NPV) and positive predictive value (PPV) of RT-LAMP for detection of Pfs16 transcript were 100% and 96.8%, respectively, and 100% for both when employing Pfs25 transcript. Detection rate of Pfs16 and Pfs25 transcripts by RT-LAMP in microscopically gametocyte-negative samples was 91.7% and 29.2%, respectively. Compared with nested RT-PCR, RT-LAMP had a higher sensitivity but similar specificity, with the advantage of a shorter assay time. As RT-LAMP requires very basic instruments and the results can be obtained by visual inspection, this technique provides a simple and reliable tool for epidemiological studies of malaria transmission and in gametocyte-targeted control programmes.