Abstract Background We recently demonstrated that the measurement of Mycoplasma pneumoniae (Mp)-specific immunoglobulin (Ig)M antibody-secreting cells (ASCs) improved diagnosis of Mp infection. Here, we aimed to describe Mp ASC kinetics and duration in comparison to conventional measures such as pharyngeal Mp deoxyribonucleic acid (DNA) and serum antibodies. Methods This is a prospective longitudinal study of 63 community-acquired pneumonia (CAP) patients and 21 healthy controls (HCs), 3–18 years of age, from 2016 to 2017. Mycoplasma pneumoniae ASCs measured by enzyme-linked immunospot assay were assessed alongside Mp DNA and antibodies during 6-month follow-up. Results Mycoplasma pneumoniae ASCs of the isotype IgM were found in 29 (46%), IgG were found in 27 (43%), and IgA were found in 27 (43%) CAP patients. Mycoplasma pneumoniae ASCs were detected from 2 days to a maximum of 6 weeks after symptom onset, whereas Mp DNA and antibodies persisted until 4 months (P = .03) and 6 months (P < .01). Mycoplasma pneumoniae ASCs were undetectable in HCs, in contrast to detection of Mp DNA in 10 (48%) or antibodies in 6 (29%) controls for a prolonged time. The Mp ASC response correlated with clinical disease, but it did not differ between patients treated with or without antibiotics against Mp. Conclusions Mycoplasma pneumoniae-specific ASCs are short-lived and associated with clinical disease, making it an optimal resource for determining Mp pneumonia etiology. Mycoplasma pneumoniae (Mp)-specific antibody-secreting cells (ASCs) are detectable in peripheral blood of children with Mp community-acquired pneumonia within the first 1–2 weeks upon disease onset, and its presence is short-lived and associated with clinical disease.