Emerging evidence shows that desaturase 5 (Δ5), the key regulator in the synthesis of highly unsaturated long-chain fatty acids (HUFA), is modulated by factors including adiposity, diet and insulin resistance. We explored the association of these factors in a cross-sectional study within a high-risk Cree population. Anthropometric measures and fasting blood glucose and insulin were analysed. Δ5 was estimated as the 20 : 4n-6:20 : 3n-6 ratio in erythrocyte membranes. The setting of the present study was the Mistissini community in the Cree Territory of Québec, Canada with ninety-eight female and sixty-eight male subjects aged 20–88 years. Obesity (BMI ≥ 30 kg/m2) was prevalent across age groups. Δ5 was inversely associated with BMI (Spearman's correlation coefficient (rs) − 0·175; P = 0·03) and positively associated with age (rs 0·593; P < 0·0001), which was driven by age-related increases in dietary intake of n-3 fatty acids and decreases in 20 : 3n-6. Homeostasis model assessment of insulin resistance (HOMA-IR) was significantly inversely associated with Δ5 in age-adjusted linear regression analyses in normoglycaemic individuals (β − 2·110 (se 0·566); P < 0·001), whereas no association was observed among glucose-intolerant individuals (interaction term P = 0·03). In contrast, there were no significant interactions indicating differences in the slope for each of the adiposity measures in their associations with Δ5. The present study indicates that the dietary transition of reduced consumption of fish among younger Cree may compound the effects of obesity and emerging insulin resistance which, in turn, could reduce bioavailability of HUFA n-3 (through reduced Δ5 activity). Also, the study suggests that disease progression is an important consideration when evaluating correlates of Δ5 activity in observational studies.