Choline plays many important roles, including the synthesis of acetylcholine, and may affect muscle responses to exercise. We previously observed correlations between low choline intake and reduced gains in strength and lean mass following a 12-week resistance exercise training (RET) program for older adults. To further explore these findings, we conducted a randomized controlled trial. Three groups of 50-to-69-year-old healthy adults underwent a 12-week RET program (3x/week, 3 sets, 8–12 reps, 70% of maximum strength (1RM)) and submitted >48 diet logs (>4x/week for 12 weeks). Participants’ diets were supplemented with 0.7 mg/kg lean/d (low, n = 13), 2.8 mg/kg lean/d (med, n = 11), or 7.5 mg/kg lean/d (high, n = 13) of choline from egg yolk and protein powder. The ANCOVA tests showed that low choline intake, compared with med or high choline intakes, resulted in significantly diminished gains in composite strength (leg press + chest press 1RM; low, 19.4 ± 8.2%; med, 46.8 ± 8.9%; high, 47.4 ± 8.1%; p = 0.034) and thigh-muscle quality (leg press 1RM/thigh lean mass; low, 12.3 ± 9.6%; med/high, 46.4 ± 7.0%; p = 0.010) after controlling for lean mass, protein, betaine, and vitamin B12. These data suggest that low choline intake may negatively affect strength gains with RET in older adults.