Isotopic analysis of serially sampled vibrissae (whiskers) is a powerful method to investigate changes in an individual's resource and habitat use over time, which is difficult or impossible to accomplish using traditional dietary proxies such as observation or scat analysis. A vibrissae-based isotopic approach is limited by knowledge of vibrissae growth rates, which are required to determine the time period represented by each subsampled segment. Likewise, determining the magnitude of, and variation in, isotopic differences between a consumer and its diet, commonly referred to as trophic discrimination factors (TDFs), is a crucial step in quantifying diet composition using stable isotopes. TDF estimates are available only for a few mammalian taxa. We measured vibrissae growth rates and δ¹³C and δ¹⁵N TDFs in captive southern sea otters (Enhydra lutris nereis). Sea otters were administered ¹⁵N-enriched glycine intravenously and vibrissae were collected periodically and serially sampled for carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope analysis. Growth of adult sea otter vibrissae was linear with a mean (±SD) rate of 7.7 (± 1.2) cm/year. Mean (±SD) whole diet–vibrissae TDFs were 2.8‰ (±0.2‰) for δ¹³C and 5.5‰ (±0.2‰) for δ¹⁵N. Mean (±SD) lipid-extracted diet–vibrissae TDFs were 2.4‰ (±0.2‰) for δ¹³C and 4.9‰ (±0.3‰) for δ¹⁵N. δ¹³C TDFs were similar to previously reported values for mammalian carnivores, but δ¹⁵N TDFs were higher than expected. These results will increase the accuracy of isotopic diet analyses of mustelids and other carnivores for which there are few estimates of TDFs and no estimates of vibrissae growth rates.