Background: This study aimed to investigate the effect of 6 weeks’ pilates training along with dill supplementation on serum concentrations of nesfatin-1, lipocaline-2, and insulin resistance in females with overweight and obesity. Materials and Methods: In this randomized controlled trial study, 45 overweight and obese females are randomly assigned to four groups: Pilates training + dill group (PDG) ( n = 12), pilates training + placebo group (PPG) ( n = 11), dill supplementation group (DG) ( n = 11), and placebo group (PG) ( n = 11). Participants of PDG and PPG performed pilates training for 6 weeks (60 min, 3 sessions per week). PDG and DG received dill tablet (three times a day, 6 weeks). Anthropometric measurements, glycemic markers, and blood samples were assessed before (pretest) and after (posttest) 6 weeks of intervention. Results: Results showed a significant increase in serum concentrations of nesfatin-1 in PDG compared to pretest ( P = 0.001). Differences in the serum concentrations of nesfatin-1 in PDG were greater than PPG, DG, and PG ( P = 0.01). Furthermore, results found in significant reduction in serum concentrations of lipocalin-2, body mass index (BMI), and waist-hip ratio (WHR) in PDG, PPG, and DG as compared to pretest ( P > 0.05). Fasting glucose plasma (FGP) was significantly decreased in all three intervention groups PDG ( P < 0.001), PPG ( P < 0.001), and DG ( P < 0.001) as compared to pretest. Differences in FGP were significantly higher in PDG than PPG, DG, and PG ( P = 0.001). A significant reduction was found for insulin only in PDG after 6 weeks of intervention as compared to pretest ( P = 0.03). Insulin resistance significantly decreased in PDG ( P = 0.03) and PPG ( P = 0.04) as compared to pretest. Body fat percent (BFP) was significantly decreased in PDG ( P = 0.003), PPG ( P = 0.006), and DG ( P = 0.01). However, there were no significant inter-group differences in insulin resistance, insulin, serum concentrations of lipocalin-2, BMI, BFP, and WHR after 6 weeks of Pilates training along with dill supplementation ( P > 0.05). Conclusion: We concluded that 6 weeks of Pilates training along with dill may be beneficial for improvements in serum concentrations of nesfatin-1 and FGP.