The objective of this meta-analysis was to investigate the effects of plant-derived polyunsaturated fatty acids (PUFAs) on glucose metabolism and insulin resistance. Scopus and PubMed databases were searched until January 2018. Eligible studies were randomized controlled feeding trials that investigated the effects of a diet high in plant-derived PUFA as compared with saturated fatty acids (SFA) or carbohydrates and measured markers of glucose metabolism and insulin resistance as outcomes. Data from 13 relevant studies (19 comparisons of plant-derived PUFA with control) were retrieved. Plant-derived PUFA did not significantly affect fasting glucose (−0.01 mmol/L (95 % CI − 0.06 to 0.03 mmol/L)), but lowered fasting insulin by 2.6 pmol/L (−4.9 to −0.2 pmol/L) and homeostatic model assessment-insulin resistance (HOMA-IR) by 0.12 units (-0.23 to − 0.01 units). In dose–response analyses, a 5% increase in energy (En%) from PUFA significantly reduced insulin by 5.8 pmol/L (95% CI −10.2 to −1.3 pmol/L), but not glucose (change −0.07, 95% CI −0.17 to 0.04 mmol/L) and HOMA-IR (change − 0.24, 95% CI −0.56 to 0.07 units). In subgroup analyses, studies with higher PUFA dose (upper tertiles) reduced insulin (-6.7, –10.5 to −2.9 pmol/L) and HOMA-IR (-0.28, –0.45 to −0.12 units), but not glucose (−0.09, 95% CI −0.18 to 0.01 mmol/L), as compared with an isocaloric control. Subgroup analyses showed no differences in effects between SFA and carbohydrates as replacement nutrients (p interaction ≥0.05). Evidence from randomized controlled trials indicated that plant-derived PUFA as an isocaloric replacement for SFA or carbohydrates probably reduces fasting insulin and HOMA-IR in populations without diabetes.