Spexin (SPX) is a novel peptide thought to have a role in various metabolic regulations. Given its presumed body-weight regulatory functions, we aimed to determine whether lifestyle intervention programs on weight loss and fasting glucose (FG) improvement among people with impaired glucose regulation also alter levels of circulating SPX. A total of 160 Saudi adult males and females with prediabetes were randomly selected from a larger cohort (N = 294) who underwent a 6-month lifestyle modification program to improve their glycemic status. Participants were split into two groups based on differences in glucose levels post-intervention, with the first 50% (improved group) having the most significant reduction in FG. SPX was measured at baseline and after 6 months. Changes in SPX was significant only in the improved group [baseline: median (Q1-Q3) of 164 pg/ml (136-227) vs follow-up: 176 pg/ml (146-285); p < 0.01]. When stratified by sex, the significant increase was observed only in females [159 pg/ml (127-252) vs 182.5 (152,369.1); p < 0.01]. Furthermore, SPX levels showed a significant inverse association with FG (β = -0.22, p = 0.003) even after adjustment with age and BMI, again only in females. Circulating SPX levels increase over time in people with prediabetes, particularly women who responded favorably in a 6-month lifestyle intervention program. Whether an unknown mechanism regulating the sexual disparity seen in SPX levels post-intervention exists should be further investigated using a larger sample size.