Human serum albumin (HSA) has been investigated as a serum half-life extender of therapeutic proteins thanks to its unusually long serum half-life. However, in mice, the serum half-life of a HSA-conjugated protein was much shorter than that of HSA in humans, likely due to the species-dependent nature of albumin-FcRn interactions. Herein, we investigated species-dependent albumin-FcRn interactions using species-matched albumin (mouse serum albumin) and species-mismatched albumin (HSA) in non-transgenic mice. We site-specifically introduced a clickable non-natural amino acid to a target protein followed by conjugation to an albumin species via a hetero-bifunctional linker. Using in vitro binding assays, we showed that both HSA- and MSA-conjugated proteins bound mouse FcRns. Conjugation of HSA led to very limited extension of the serum half-life of sfGFP in mice (16.3 h), compared to that of HSA in transgenic mice harboring an allele of mouse FcRn knock-out and expressing humn FcRn (67 h) reported previously. These results suggest that the FcRn-mediated recycling of HSA is not effective in mice. However, conjugation of mouse serum albumin (MSA) resulted in a serum half-life of sfGFP (27.7 h) comparable to that of MSA in mice (28.8 h). Altogether, our study supported that albumin-FcRn interactions are species dependent in vivo.