Men have been reported to experience greater increases in muscle protein synthesis and mitochondrial biogenesis, and indices of glycemic control, after several weeks of sprint interval training (SIT) as compared to women. The potential impact of sex on the skeletal muscle response to an acute session of SIT has not been previously examined and could provide a potential mechanistic framework to explain potential divergent training adaptations. The present study examined the acute response of genes involved in skeletal muscle metabolism and structural remodelling to a single session of SIT in men and women matched for initial fitness [n = 8/8, age = 23±4/22±3 y, peak aerobic capacity (VO2peak) = 45±6/45±10 ml/kg fat free mass/min]. None of the women were using oral contraceptives and all were tested in the mid-follicular phase of their menstrual cycles (day 9±2). Subjects completed a session of SIT consisting of 3 x 20-sec all-out cycling efforts against a resistance of 5% body mass, interspersed with 2 min of recovery. Biopsies from m. vastus lateralis were obtained before and immediately and 3 h after exercise. Gene expression was determined using real-time quantitative polymerase chain reaction and analyzed using a two-factor ANOVA (sex, time). Women had higher expression of hexokinase II (HK2) and lower expression of forkhead box O3 (FOXO3) compared to men (main effects for sex, p<0.05). Exercise increased the mRNA expression of FOXO3, HK2, hormone sensitive lipase (LIPE), muscle ring-finger protein-1 (TRIM63), myogenic differentiation 1 (MYOD1), peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A), pyruvate dehydrogenase kinase isozyme 4 (PDK4) and vascular endothelial growth factor A (VEGFA) at 3 h vs rest (all main effects, p<0.05). Women had lower expression of glucose transporter 4 (SLC2A4) at rest but showed an exercise-induced increase in the expression of SLC2A4 at 3 h. Exercise also increased the expression of lipoprotein lipase (LPL) after 3 h of recovery in women only (p<0.05); whereas, the exercise-induced increase in the expression of Atrogin-1 (FBXO32) was greater in men vs women (p<0.05). There was no effect of exercise or sex on the expression of glycogen synthase kinase-3α (GSK3A) or insulin-like growth factor (IGF1) (p>0.05). In summary, the gene expression response to an acute bout of SIT was generally similar between men and women. The metabolic basis for reported sex-based differences in specific training responses remains to be elucidated. These data also demonstrate that as little as 1 min of intense intermittent exercise alters the expression of genes involved in skeletal muscle metabolism and structural remodeling.