Yeasts are the most predominant petroleum hydrocarbon-degrading fungi isolated from petroleum-contaminated soil. However, information of the transmembrane transport of petroleum hydrocarbon into yeast cells is limited. The present study was designed to explore the transmembrane transport mechanisms of the typical petroleum hydrocarbon n-hexadecane in Candida tropicalis cells with petroleum hydrocarbon biodegradation potential. Yeast cells were treated with n-hexadecane in different scenarios, and the percentage of intracellular n-hexadecane and transport dynamics were investigated accordingly. The intracellular concentration of n-hexadecane increased within 15 min, and transportation was inhibited by NaN3, an ATPase inhibitor. The uptake kinetics of n-hexadecane were well fitted by the Michaelis-Menten model, and Kt values ranged from 152.49 to 194.93 mg/L. All these findings indicated that n-hexadecane might cross the yeast cells in an energy-dependent manner and exhibit an affinity with the cell transport system. Moreover, the differentially expressed membrane proteins induced by n-hexadecane were identified and quantified by tandem mass tag labeling coupled with liquid chromatography tandem mass spectrometry analysis. The proteome analysis results demonstrated that energy production and conversion accounted for a large proportion of the functional classifications of the differentially expressed proteins, providing further evidence that sufficient energy supply is essential for transmembrane transport. Protein functional analysis also suggested that differentially expressed proteins associated with transmembrane transport processes are clearly enriched in endocytosis and phagosome pathways (p < 0.05), and the analysis supported the notion that the underlying transmembrane transport mechanism might be associated with endocytosis and phagosome pathways, revealing a new mechanism of n-hexadecane internalization by Candida tropicalis.