In cold marine environments, the obligate hydrocarbon‐degrading psychrophile Oleispira antarctica RB‐8, which utilizes aliphatic alkanes almost exclusively as substrates, dominates microbial communities following oil spills. In this study, LC–MS/MS shotgun proteomics was used to identify changes in the proteome induced during growth on n‐alkanes and in cold temperatures. Specifically, proteins with significantly higher relative abundance during growth on tetradecane (n‐C14) at 16°C and 4°C have been quantified. During growth on n‐C14, O. antarctica expressed a complete pathway for the terminal oxidation of n‐alkanes including two alkane monooxygenases, two alcohol dehydrogenases, two aldehyde dehydrogenases, a fatty‐acid‐CoA ligase, a fatty acid desaturase and associated oxidoreductases. Increased biosynthesis of these proteins ranged from 3‐ to 21‐fold compared with growth on a non‐hydrocarbon control. This study also highlights mechanisms O. antarctica may utilize to provide it with ecological competitiveness at low temperatures. This was evidenced by an increase in spectral counts for proteins involved in flagella structure/output to overcome higher viscosity, flagella rotation to accumulate cells and proline metabolism to counteract oxidative stress, during growth at 4°C compared with 16°C. Such species‐specific understanding of the physiology during hydrocarbon degradation can be important for parameterizing models that predict the fate of marine oil spills.