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The aim was to investigate the associations between different physical activity (PA) patterns and sedentary time (ST) with vitamin D deficiency (<12 ng/mL) in a large sample of Chilean women. In this cross-sectional study, the final sample included 1245 adult and 686 older women. The PA levels, mode of commuting, ST, and leisure-time PA were self-reported. Vitamin D deficiency was defined as <12 ng/mL and insufficiency as <20 ng/mL. A higher ST was associated with vitamin D deficiency (odds ratio (OR): 2.4, 95%: 1.6⁻4.3) in adults, and passive commuting was associated with vitamin D deficiency in older (OR: 1.7, 95%: 1.1⁻2.7). Additionally, we found a joint association in the high ST/passive commuting group in adults (OR: 2.8, 95%: 1.6⁻4.9) and older (OR: 2.8, 95%: 1.5⁻5.2) with vitamin D deficiency, in respect to low ST/active commuting. The PA levels and leisure-time PA were not associated with vitamin D deficiency. In conclusion, mode of commuting and ST seems important variables related to vitamin D deficiency. Promoting a healthy lifestyle appears important also for vitamin D levels in adult and older women. Further studies are needed to establish causality of this association and the effect of vitamin D deficiency in different diseases in this population.