Objective This study investigated whether the levels of specific serum microRNAs (miRNAs) were altered following diet‐induced weight loss and whether the serum miRNAs differed in the presence of the metabolic syndrome. Methods The study was a weight loss intervention trial with a prescribed energy deficit of approximately 500 kcal/d. Levels of 22 miRNAs were determined in serum samples from 85 participants with overweight or obesity. miRNAs were analyzed using TaqMan Array miRNA Cards and normalized to the geometric mean of spiked‐in ath‐miR‐159a and U6 small nuclear RNA using the ΔCT method. Results The average weight loss was 5.7 kg (P < 0.001). miR‐122‐5p (−0.18 ± 0.06 log fold relative to initial, P < 0.01) and miR‐193a‐5p (−0.12 ± 0.04, P < 0.01) levels decreased in response to weight loss. miR‐126a‐3p (0.11 ± 0.04, P = 0.01) and miR‐222‐3p (1.51 ± 0.12, P < 0.001) levels increased. Furthermore, a higher level of miR‐122‐5p was observed at baseline in participants with the metabolic syndrome compared with participants without (0.28 ± 0.08, P < 0.01). Conclusions Changes in circulating miR‐122‐5p, miR‐126a‐3p, miR‐193a‐5p, and miR‐222‐3p in response to diet‐induced weight loss are demonstrated. Furthermore, assessment of miR‐122‐5p could be an indicator of an adverse metabolic health status independent of obesity.