Trans fatty acids in human milk have raised concerns because of possible adverse effects on infant growth and development. Analyses of human milk in the late 1990s in Canada showed high amounts of trans fatty acids from partially hydrogenated oils. Canada introduced labeling of trans fatty acids on retail foods in 2003. We analyzed trans and cis unsaturated and saturated fatty acids in human milk collected from 87 women in 2004–2006 and compared the levels to those in milk collected from 103 women in 1998 and analyzed using similar methods. The total trans fatty acids (mean ± SEM, g/100 g fatty acids) in human milk in Canada decreased significantly, from 7.1 ± 0.32 in 1998 to 6.2 ± 0.48, 5.3 ± 0.49, and 4.6 ± 0.32 over 3 consecutive 5-mo periods from November 2004 to January 2006. The milk total trans fatty acids were significantly and inversely related to 16:0, 18:2(n-6), 18:3(n-3), 20:4(n-6), 22:4(n-6), and 22:5(n-6) and positively related to 18:0 and conjugated linolenic acids (P < 0.05, n = 190). The estimated exposures of exclusively breast-fed infants to trans fatty acids decreased from a mean and 95th percentile intake of 2.0 and 4.4 g · infant−1 · d−1 in 1998 to 1.33 and 2.41 g · infant−1 · d−1, respectively, in late 2005. The estimated intake of the mothers was 4.0 (range 0.51–12.3) and 2.2 (0.56–7.65) g · person−1 · d−1 in 1998 and late 2005, respectively. Our studies show trans fatty acids have decreased in human milk in Canada, which suggests a concomitant decrease in trans fatty acid intake among lactating women and breast-fed infants.