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Zinc inhibits vascular calcification in vivo and in vitro. Patients with type 2 diabetes mellitus show hypozincemia and are at an elevated risk of cardiovascular events. Recently, an in vitro test (T₅₀-test) was developed for determination of serum calcification propensity and a shorter T₅₀ means a higher calcification propensity. This cross-sectional study investigated the association between serum zinc and T₅₀ in 132 type 2 diabetes mellitus patients with various kidney functions. Furthermore, the effect of exogenous zinc on T₅₀ was also investigated in vitro using separately pooled serum samples obtained from healthy volunteers and patients with hemodialysis. We measured T₅₀ levels using the established nephelometric method. The median (interquartile range) levels of T₅₀ and serum zinc were 306 (269 to 332) min, and 80.0 (70.1 to 89.8) µg/dL, respectively. Serum zinc level showed a weak, but positive correlation with T₅₀ (<i>r_s</i> = 0.219, <i>p</i> = 0.012). This association remained significant in multivariable-adjusted analysis, and was independent of known factors including phosphate, calcium, and magnesium. Kidney function and glycemic control were not significantly associated with T₅₀. Finally, in vitro experiments showed that addition of a physiological concentration of exogenous zinc chloride significantly increased serum T₅₀. Our results indicate that serum zinc is an independent factor with a potential role in suppressing calcification propensity in serum.