Abstract Background/objectives Numerous hepatokines are involved in inter-organ cross talk regulating tissue-specific insulin sensitivity. Adipose tissue lipolysis represents a crucial element of adipose insulin sensitivity and is substantially involved in long-term body weight regulation after dietary weight loss. Thus, we aimed to analyze the impact of the hepatokine Fetuin-B in the context of weight loss induced short- and long-term modulation of adipose insulin sensitivity. Subjects/methods 143 subjects (age > 18; BMI ≥ 27 kg/m²) were analyzed before (T-3) and after (T0) a standardized 12-week dietary weight reduction program. Afterward, subjects were randomized to a 12-month lifestyle intervention or a control group. After 12 months (T12) no further intervention was performed until 6 months later (T18) (Maintain-Adults trial). Tissue-specific insulin sensitivity was estimated by HOMA-IR (predominantly liver), ISI_Clamp (predominantly skeletal muscle), and free fatty acid suppression during hyperinsulinemic-euglycemic clamp (FFA_Supp) (predominantly adipose tissue). Fetuin-B was measured at all concomitant time points. Results Circulating Fetuin-B levels correlated significantly with estimates of obesity, hepatic steatosis as well as HOMA-IR, ISI_Clamp, FFA_Supp at baseline. Fetuin-B decreased during dietary weight loss (4.2 (3.5–4.9) vs. 3.8 (3.2–4.6) µg/ml; p = 2.1 × 10⁻⁵). This change was associated with concomitant improvement of HOMA-IR (r = 0.222; p = 0.008) and FFA_Supp (r = −0.210; p = 0.013), suggesting a particular relationship to hepatic and adipose tissue insulin sensitivity. Weight loss induced improvements of insulin resistance were almost completely preserved until months 12 and 18 and most interestingly, the short and long-term improvement of FFA_Supp was partially predicted by baseline level of Fetuin-B. Conclusions Our data suggest that Fetuin-B might be a potential mediator of liver-adipose cross talk involved in short- and long-term regulation of adipose insulin sensitivity, especially in the context of diet-induced weight changes. Trial registration ClinicalTrials.gov number: NCT00850629, https://clinicaltrials.gov/ct2/show/NCT00850629, date of registration: February 25, 2009.