Latex of Calotropis procera has been described as a relevant source of pharmacologically active proteins, including proteins with anticancer activity. A previous in vitro study of laticifer proteins (LP) from C. procera reported that they had selective cytotoxic effects on human cancer cell lines. The aim of this study was to determine the effects of LP in vivo using mice transplanted with sarcoma 180. Biochemical, hematological, histopathological, and morphological analyses were performed in animals given LP by oral or intraperitoneal routes. LP significantly reduced tumor growth (51.83%) and augmented the survival time of animals for up to 4 days. Tumor growth inhibitory activity was lost when LP fraction was submitted to proteolysis, acidic treatment, or pretreated with iodoacetamide. However, LP retained its inhibitory activities on sarcoma 180 growth after heat treatment. Thus, it seems that heat-stable proteins are involved in tumor suppression. Biochemical parameters, such as the enzymatic activity of aspartate aminotransferase and alanine aminotransferase and urea content in serum were not affected in treated mice. It is worth noting that LP completely eliminated the 5-FU-induced depletion of leukocytes in mice even when given orally. The active proteins were recovered in a single fraction by ion exchange chromatography and still exhibited anticancer activity. This study confirms the pharmacological potential of proteins from the latex of C. procera to control sarcoma cell proliferation.