Background Betatrophin may increase islet β cell proliferation in insulin resistance and irisin may improve glucose tolerance in mice. To examine the relationship between betatrophin and irisin, we investigated it in middle-aged Chinese subjects with normal glucose tolerance (NGT) and type 2 diabetes mellitus (T2DM). Methods A total of 460 permanent residents of Fengxian District, aged 40–60 years and without T2DM, were enrolled. Anthropometric parameters, oral glucose tolerance test (OGTT) results, glycosylated haemoglobin levels, blood lipid levels, insulin sensitivity (homeostasis model assessment of insulin resistance, HOMA-IR), β cell function (homeostasis model assessment-β, HOMA-β), estimated glomerular filtration rate (eGFR) and body fat composition were determined. Matched for age, gender and body mass index (BMI, 18–28 kg/m2), newly diagnosed T2DM (n = 50, male/female = 23/27) and NGT (n = 50, male/female = 21/29) subjects were selected based on the results of an OGTT. Serum betatrophin and irisin levels were determined by enzyme linked immune sorbent assay (ELISA). Results Males had higher levels of betatrophin compared with females in both the NGT and T2DM groups. Compared with NGT subjects, the level of betatrophin in the T2DM group was higher, and males in the T2DM group had higher betatrophin levels than males in the NGT group, but there was no significant difference in betatrophin levels in females between the T2DM and NGT groups. Spearman’s correlation analysis revealed that serum betatrophin levels in females with NGT were positively correlated with irisin and FINS (fasting insulin) levels ( p < 0.05), but no correlation was found between betatrophin and irisin levels in males with NGT or in males or females with T2DM. In females with T2DM, circulating betatrophin levels were positively correlated with weight, BMI and hip circumference (p < 0.05) but negatively correlated with FPG (fasting plasma glucose) and HOMA-IR (p < 0.05). Conclusions Gender differences in the relationship between betatrophin and irisin indicate that there might be cytokine-mediated crosstalk among the liver, adipose tissue and skeletal muscle.