Pups born to mice with a targeted deletion of relaxin or its receptor (Rxfp1) die within 24 h postpartum. This has been attributed, in part, to abnormal mammary gland development in relaxin-mutant mice (Rln–/–). However, mammary development is normal in relaxin receptor-mutant (Rxfp1–/–) mice. The present study aimed to verify the mammary phenotypes in late pregnant and early lactating Rln–/– mice and to test the hypothesis that relaxin is involved in milk protein synthesis. Comparisons between late pregnant and early lactating wildtype (Rln+/+) and Rln–/– mice showed no differences in lobuloalveolar structure or ductal branching in the mammary gland. Mammary explants from Rln–/– mice also expressed β-casein and α-lactalbumin in response to lactogenic hormones at a similar level to Rln+/+ mice, implying normal milk protein synthesis. Pregnant Rln–/– mice infused with relaxin for 6 days gave birth to live pups without difficulty, and 96% of pups survived beyond 7 days. This is in contrast with the 100% pup mortality in saline-treated Rln–/– mice or 3-day relaxin-treated Rln–/– mice. Pups born to relaxin-treated Rln–/– dams weighed significantly less than Rln+/+ pups but had similar growth rates as their wildtype counterparts. In summary, relaxin is not critical for mammary gland development or β-casein and α-lactalbumin expression in late pregnant mice. In addition, Rln–/– dams did not need to be treated with relaxin postpartum for the pups to survive, suggesting that relaxin has no role in the maintenance of lactation in mice.