Context: Impaired sensitivity to the antilipolytic action of insulin in adipose tissue (AT) may play a role in determining metabolic dysfunction in polycystic ovary syndrome (PCOS). Objectives: To test the hypothesis that insulin resistance (IR) in AT is associated with whole-body insulin sensitivity and β-cell function in PCOS. Research Design and Setting: Prospective cross-sectional study. Methods: Eighteen participants with PCOS and 18-matched control participants underwent a modified frequently sampled intravenous glucose tolerance test (mFSIVGTT); subgroups underwent single-slice computed tomography scans determining AT distribution and adipocyte glucose transporter type 4 (GLUT-4) expression. Main Outcome Measures: IR in AT in basal (by the adipose insulin resistance index [Adipo-IR]) and dynamic (mFSIVGTT-derived indices of insulin-mediated nonesterified fatty acids [NEFA] suppression [NEFAnadir, TIMEnadir, and %NEFAsupp]) states; whole-body insulin-mediated glucose uptake and insulin secretion in basal (by homeostatic model assessment [HOMA]-IR and HOMA- β%) and dynamic (mFSIVGTT-derived insulin sensitivity index [Si], acute insulin response to glucose [AIRg], and disposition index [Di]) states. Results: Participants with PCOS had higher HOMA-IR and HOMA-β%, lower Si and Di, higher longer TIMEnadir, higher Adipo-IR and NEFAnadir, and a trend toward lower GLUT-4, than the control group participants. Adipo-IR was associated with dynamic state IR in AT (NEFAnadir TIMEnadir, and %NEFAsupp), but only in PCOS, and with HOMA-IR and HOMA-β% in both groups. NEFAnadirand TIMEnadirwere negatively and %NEFAsupppositively associated with Si only in PCOS, but not with AIRg and Di, or GLUT-4 expression. Conclusion: Women with PCOS demonstrated increased IR in AT, which is closely associated with whole-body IR but not with dynamic state β-cell function or adipocyte GLUT-4 gene expression.