The Na+,K+-ATPase (NKA) is a key protein involved in the maintenance of skeletal muscle excitability and contractility, and is expressed as multiple isoforms for catalytic (α1-3) and regulatory (β1-3) subunits. Increases in muscle NKA are established for endurance and sprint training, but the effects of resistance exercise training are less clear. We investigated whether muscle [3H]ouabain binding site content, which measures NKA content, was increased after resistance training and which NKA isoforms (α1-α3, and β1-β3) accounted for any NKA upregulation. Twenty-one healthy, young adult males participated in the study (age, 22.9 ± 4.6 yr; height, 1.8 ± 0.70 m, mass: 85.1 ± 17.8 kg, mean ±SD) and underwent either 7 weeks of either resistance training (RT, n=16) or control (CON, n=5). Participants performed three sets of 10-12 repetitions of each exercise interspersed with 60 – 90 s of recovery between sets. To induce progressive overload, the training weight was incremented by ~5% when a participant completed 12 repetitions of the three total sets. The training program involved upper and lower body exercises and comprised back squat, leg raise, leg press, bench press, incline bench press, lat pulldown, lunge, dumbbell shoulder press, dumbbell bicep curl, lying triceps extensions, bent-over row, upright row, barbell biceps curl, dips and sit-ups. The training was conducted in a laboratory under supervision. Participants underwent a resting vastus lateralis biopsy prior to and following RT or CON, for measurement of NKA content and NKA α1-3 and β1-3 isoform abundances. The one repetition maximum (1RM) leg press increased after RT from 339 ±79 kg to 465 ±111 kg (37%, p=0.001) whilst the 1RM bench press increased from 80 ±17 kg to 86 ±21 kg (8%, p=0.01). The muscle NKA content increased (student t-test) by 12% after RT (311 ±76 vs 349 ±76 pmol.g wet weight-1, p = 0.012), with no change in CON (316 ±86 vs 328 ±70 pmol.g wet weight-1, p=0.271). The NKA α1 isoform increased by 32% (p=0.013) and the α2 isoform increased by ~10% after RT (p=0.001), with no significant changes in CON (p=0.174 and p=0.33, respectively). These increases in each of NKA content, α1 and α2 isoform abundances with RT were robust, observed in 14, 14 and 16 of the participants, respectively. However, no differences were found in the β1 or β2 isoform abundances following RT (p=0.180 and p=0.221, respectively). The α3 and β3 isoforms were not detected. Thus, an increase in muscle NKA content induced by resistance training comprised increases in both of the α1 and α2 NKA isoform abundances. The increased NKA α1 may facilitate recovery of Na+/K+ disturbances after sets within training sessions, and increases in NKA α2 could contribute to minimising Na+/K+ disturbances and thereby resisting fatigue during intense resistance training sessions.