Omission of the dry period of cows improved energy balance (EB) and showed variable effects on somatic cell counts (SCC) and natural antibodies (NAb) in milk. A glucogenic diet compared with lipogenic diet enhanced plasma NAb binding keyhole limpet hemocyanin (KLH). NAb in milk were associated with SCC. It was hypothesized that during negative energy balance, NAb either have a role or reflect in inflammatory processes and are associated with SCC. The objective was to study effects of dry period length and dietary energy source on titers of NAb binding KLH and lipopolysaccharide (LPS) in milk, SCC and mastitis. In total, 167 Holstein-Friesian dairy cows were randomly assigned to treatments. Treatments consisted of 3 dry period lengths: 0-, 30- or 60-d and 2 early lactation diets (glucogenic or lipogenic), in a 3 × 2 factorial design. Cows enrolled in this study were clinically healthy and had SCC in milk < 250,000 cell/mL. Milk samples for NAb and SCC measurement were sampled weekly and 4 times per week, respectively, from wk 1 until 14 postpartum. The data collected were statistically analyzed using ANOVA and logistic regression. Cows with a 0-d dry period had higher titers of IgG and IgM binding KLH and LPS and higher SCC in milk compared with cows with a 30- or 60-d dry period (P < 0.01). Mastitis incidence was 17% and did not differ between dry period lengths or lactation diets. A glucogenic diet showed higher titers of IgM binding LPS and tended to have higher titers of IgG binding LPS in milk compared with a lipogenic diet (P < 0.01 and P = 0.08, respectively). Higher titers of IgG and IgM binding KLH and IgG binding LPS were associated with increased risk of high SCC (P < 0.05). Higher IgG and IgM binding KLH and LPS were also associated with increased risk of mastitis (P < 0.05). The results demonstrate that cows with a 0-d dry period and fed a glucogenic diet showed high titers of IgG binding LPS in milk. Moreover, we can conclude that IgG and IgM binding KLH or LPS in milk might be additional valuable tools to detect increased risks for mastitis in dairy cows.