Context: Puberty is initiated by a complex interaction of suppressing and stimulating factors. Genetic studies of familial central precocious puberty have suggested makorin RING-finger protein 3 (MKRN3) as a major inhibitor of GnRH secretion during childhood. Furthermore, genetic variation near MKRN3 (rs12148769) affects age at menarche in healthy girls. Objective: To evaluate if serum levels of MKRN3 declined prior to pubertal onset in healthy girls. Design: 1) Population-based longitudinal study of healthy Danish girls; 2) Cohort study of early maturing girls. Setting: 1) General community; 2) Tertiary referral centre for pediatric endocrinology. Patients or Other Participants: Healthy girls (n=38) aged 9.3 (5.9-11.3) years at baseline followed for 6.0 (2.7-7.6) years (2006-2014) with blood sampling every 6 months. Early maturing girls (n=13) with breast development < 8.3 years of age. Interventions: None Main outcome Measures: Serum levels of MKRN3: 354 samples (median 9, range 2 - 14 per girl). Genotyping of variants near MKRN3 (rs12148769 and rs12439354). Results: MKRN3 concentrations declined preceding pubertal onset; geometric mean (95% CI) 3 years prior to pubertal onset vs. last visit before pubertal onset: 304 (264-350) vs. 257 (243-273) pg/mL, corresponding to a reduction of 15% (1-27%) (p=0.033). In prepubertal girls, circulating MKRN3 correlated negatively with gonadotropin levels; FSH: r = -0.262 (p=0.015) and LH: r = -0.226 (p=0.037). After adjustment, MKRN3 levels were lower in early maturing girls compared to age-matched prepubertal girls: 171 (< 25-333) vs. 262 (94-624) pg/mL, p=0.051. Genetic variants near MKRN3 did not correlate with serum levels of MKRN3. Conclusions: Declining levels of circulating MKRN3 preceded pubertal onset. The negative correlation between MKRN3 and gonadotropins further support MKRN3 as a major regulator of hypothalamic GnRH secretion during childhood. Undetectable or low MKRN3 levels were observed in a subgroup of patients with early onset of puberty.