Objective: The aim of this study was to assess the role of vitamin D in cancer development in postmenopausal osteoporotic women. Methods: A cross-sectional and in vitro study was carried out, with statistical analysis with odds ratios and 95% CIs presented. Human estrogen receptor-positive breast cancer cells (MCF-7) were studied in vitro. The apoptosis-to-proliferation (A/P) ratio was also determined. Results: A total of 885 women were included in this study. Any kind of cancer was found in 112 (12.7%) of all women. Breast cancer was the most prevalent malignancy, representing half of the cases (n = 56, 50%). The prevalence of any kind of cancer and breast cancer in women with low 25-hydroxyvitamin D(3) levels (25OHD; <50 nmol/L) was higher than in women with high 25OHD levels (>= 50 nmol/L). The in vitro study demonstrated a statistically significant increased A/P ratio of 5.27 (95% CI, 4.054-6.493) with a high concentration of 1 alpha, 25-dihydroxyvitamin D (10 mu M) after 96 hours. Conclusions: Osteoporotic women with low serum levels of 25OHD (<50 nmol/L) have an increased prevalence of any kind of cancer and breast cancer; however, these differences are not statistically significant. 1 alpha, 25-dihydroxyvitamin D induced an increased A/P ratio in MCF-7 breast cancer cells in vitro.