A 2007 report by the International Agency for Research on Cancer classified night-shift work as possibly carcinogenic to humans, emphasizing, in particular, its association with breast cancer. Since this report and the publication of the last systematic review on this topic, several new studies have examined this association. Hence, to provide a comprehensive update on this topic, we performed a systematic review and meta-analysis. We searched Medline, Embase, CINAHL, Web of Science (Conference Proceedings), and ProQuest dissertations for studies published before March 1, 2012, along with a manual search of articles that cited or referenced the included studies. Included were observational case-control or cohort studies examining the association between night-shift work and breast carcinogenesis in women, which all ascertained and quantified night-shift work exposure. The search yielded 15 eligible studies for inclusion in the systematic review and meta-analysis. Using random-effects models, the pooled relative risk (RR) and 95 % confidence intervals (CIs) of breast cancer for individuals with ever night-shift work exposure was 1.21 (95 % CI, 1.00-1.47, p = 0.056, I (2) = 76 %), for short-term night-shift workers (<8 years) was 1.13 (95 % CI, 0.97-1.32, p = 0.11, I (2) = 79 %), and for long-term night-shift workers (≥8 years) was 1.04 (95 % CI, 0.92-1.18, p = 0.51, I (2) = 55 %), with substantial between-study heterogeneity observed in all analyses. Subgroup analyses suggested that flight attendants with international or overnight work exposure and nurses working night-shifts long-term were at increased risk of breast cancer, however, these findings were limited by unmeasured confounding. Overall, given substantial heterogeneity observed between studies in this meta-analysis, we conclude there is weak evidence to support previous reports that night-shift work is associated with increased breast cancer risk.