Benzylpenicilloic acid (BPNLA) is a major cleavage product of benzylpenicillin G (BP) generated after heating treatment. It is found in animal derived products from the unstable residual penicillin. Previous studies have only reported the allergic reaction caused by BPNLA, but not described its toxicity. In this study, the toxicity of BPNLA was evaluated to report the potential public health risk posed by animal derived products using in vivo and in vitro models, including the acute toxicity assays, cytotoxicity assays, apoptosis assays and cell cycle progression assay. The LD50 value for BPNLA was, 8.48 g/kg [bw] intraperitoneally. BPNLA showed cytotoxicity and inhibition of cell proliferation on SK-N-SH cells, MRC-5 cells and GC-1 cells. Further, Annexin-v/PI staining and Hoechst 33342 staining showed increased cell apoptosis and nucleus morphological changes with toxic levels of BPNLA. BPNLA arrested cells in G1 phase and reduced cells population in S phase in a dose-dependent manner. This work suggests that BPNLA might be a potential toxic agent and might have public health significance. However, the toxic concentrations of BPNLA are relatively high compared to levels that would result from the degradation of antibiotics residues in meat from animals that have received a therapeutic dose of benzylpenicillin.